Methods for treating viral infections using antibodies to aminophospholipids

ABSTRACT

Disclosed are surprising discoveries concerning the role of anionic phospholipids and aminophospholipids in tumor vasculature and in viral entry and spread, and compositions and methods for utilizing these findings in the treatment of cancer and viral infections. Also disclosed are advantageous antibody, immunoconjugate and duramycin-based compositions and combinations that bind and inhibit anionic phospholipids and aminophospholipids, for use in the safe and effective treatment of cancer, viral infections and related diseases.

[0001] The present application claims priority to co-pending U.S.application Ser. No. 10/621,269, filed Jul. 15, 2003, which claimspriority to U.S. provisional application Serial No. 60/396,263, filedJul. 15, 2002, the disclosures of which applications, including thespecification, claims, drawings and sequences, are specificallyincorporated herein by reference without disclaimer.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to the fields of aminophospholipidand anionic phospholipid biology, tumor blood vessels and viralinfections. It provides surprising new compositions, methods andcombinations for tumor vasculature targeting and cancer treatment, forinhibiting viral entry and spread and for treating viral infections. Theinvention further provides a number of preferred antibody,immunoconjugate and duramycin-based compositions that bind and inhibitaminophospholipids and anionic phospholipids for use in the treatment ofcancer, viral infections and related diseases.

[0004] 2. Description of the Related Art

[0005] Tumor cell resistance to chemotherapeutic agents represents asignificant problem in clinical oncology. Another major problem toaddress in tumor treatment is the desire for a “total cell kill”, i.e.,killing all so-called “clonogenic” malignant cells that have the abilityto grow uncontrolled and replace any tumor mass that might be removed bythe therapy. Despite certain advances in the field, these are two of themain reasons why many prevalent forms of human cancer still resisteffective chemotherapeutic intervention.

[0006] Due to the goal of developing treatments that approach a totalcell kill, certain types of tumors have been more amenable to therapythan others. For example, the soft tissue tumors, e.g., lymphomas, andtumors of the blood and blood-forming organs, e.g., leukemias, havegenerally been more responsive to chemotherapeutic therapy than havesolid tumors, such as carcinomas.

[0007] One reason for the susceptibility of soft and blood-based tumorsto chemotherapy is the greater accessibility of lymphoma and leukemiccells to chemotherapeutic intervention. Simply put, it is much moredifficult for most chemotherapeutic agents to reach all of the cells ofa solid tumor mass than it is the soft tumors and blood-based tumors,and therefore much more difficult to achieve a total cell kill.Increasing the dose of chemotherapeutic agents most often results intoxic side effects, which generally limits the effectiveness ofconventional anti-tumor agents.

[0008] Another tumor treatment strategy is the use of an “immunotoxin”,in which an anti-tumor cell antibody is used to deliver a toxin to thetumor cells. However, in common with chemotherapeutic approaches,immunotoxin therapy also suffers from significant drawbacks when appliedto solid tumors. For example, antigen-negative or antigen-deficientcells can survive and repopulate the tumor or lead to furthermetastases. A further reason for solid tumor resistance toantibody-based therapies is that the tumor mass is generally impermeableto macromolecular agents such as antibodies and immunotoxins. Both thephysical diffusion distances and the interstitial pressure within thetumor are significant limitations to this type of therapy.

[0009] An improved treatment strategy is to target the vasculature ofsolid tumors. Targeting the blood vessels of the tumors, rather than thetumor cells themselves, has certain advantages in that it is not likelyto lead to the development of resistant tumor cells, and that thetargeted cells are readily accessible. Moreover, destruction of theblood vessels leads to an amplification of the anti-tumor effect, asmany tumor cells rely on a single vessel for their oxygen and nutrients.Exemplary vascular targeting agents (VTAs) are described in U.S. Pat.Nos. 5,855,866, 5,965,132, 6,261,535, 6,051,230 and 6,451,312, whichdescribe the targeted delivery of anti-cellular agents and toxins tomarkers of tumor vasculature.

[0010] Another effective version of the vascular targeting approach isto target a coagulation factor to a marker expressed or adsorbed withinthe tumor vasculature or stroma (Huang et al., 1997; U.S. Pat. Nos.6,093,399, 6,004,555, 5,877,289, and 6,036,955). The delivery ofcoagulants, rather than toxins, to tumor vasculature has the furtheradvantages of reduced immunogenicity and even lower risk of toxic sideeffects. As disclosed in U.S. Pat. No. 5,877,289, a preferredcoagulation factor for use in such tumor-specific “coaguligands” is atruncated version of the human coagulation-inducing protein, TissueFactor (TF), the major initiator of blood coagulation.

[0011] Recently, the aminophospholipids phosphatidylserine (PS) andphosphatidylethanolamine (PE) were identified as specific markers oftumor vasculature (Ran et al., 1998). This led to the development of newanti-PS and anti-PE immunoconjugates for delivering anti-cellularagents, toxins and coagulation factors to tumor blood vessels (U.S. Pat.No. 6,312,694). In addition, it was discovered that unconjugatedantibodies to PS and PE exerted an anti-cancer effect without attachmentto a therapeutic agent, which became known as the aminophospholipid“naked antibody” approach to tumor vascular targeting and treatment(U.S. Pat. No. 6,406,693).

[0012] Although the foregoing immunoconjugate and aminophospholipidvascular targeting methods represent significant advances in tumortreatment, certain peripheral tumor cells can survive the widespreadtumor destruction caused by such therapies. Anti-angiogenic strategies,which inhibit the development of new vasculature from preexisting bloodvessels and/or circulating endothelial stem cells, are thereforecontemplated for use in combination with the VTA, coaguligand andaminophospholipid targeting methods of U.S. Pat. Nos. 5,855,866,6,093,399, 6,312,694 and 6,406,693.

[0013] Angiogenesis plays an important role in physiological processes,such as embryogenesis, wound healing and menstruation, but is alsoinvolved in certain pathological events, such as in tumor growth,arthritis, psoriasis and diabetic retinopathy (Ferrara, 1995). Asapplied to tumor treatment, anti-angiogenic strategies are based uponinhibiting the proliferation of budding vessels, generally at theperiphery of a solid tumor. These therapies are mostly applied to reducethe risk of micrometastasis or to inhibit further growth of a solidtumor after more conventional intervention (such as surgery orchemotherapy).

[0014] U.S. Pat. Nos. 6,342,219, 6,524,583, 6,342,221 and 6,416,758describe antibodies and immunoconjugates that bind to vascularendothelial growth factor-A (VEGF, formerly known as vascularpermeability factor, VPF), a primary stimulant of angiogenesis. Theseantibodies have the important advantage of inhibiting VEGF binding toonly one of the two primary VEGF receptors. By blocking VEGF binding toVEGFR2, but not VEGFR1, these antibodies have an improved safetyprofile, maintaining beneficial effects mediated via VEGFR1, e.g. inmacrophage, osteoclast and chondroclast functions.

[0015] Although the foregoing methods have advanced the art of tumortreatment, the development of additional or alternative vasculartargeting therapies is still sought. The identification of new markersof tumor vasculature is needed to expand the number of therapeuticoptions. The development of new naked antibodies with anti-cancerproperties would be a particularly important advance, as this permitsthe same targeting moiety to be used both as a single-agent therapeuticand as a vascular targeting agent for the delivery of other drugs.Therapeutic agents that have both anti-angiogenic and anti-vascular,i.e., tumor destructive, properties within the same molecule would be ofgreat value. An even more important advance would be the identificationof a class of therapeutic agents with anti-cancer properties andtherapeutic effects in other systems. The development of agents capableof treating both cancer and viral infections, two of the mostsignificant medical challenges of this era, would be a remarkable andimportant breakthrough.

SUMMARY OF THE INVENTION

[0016] The present invention addresses the foregoing and other needs ofthe prior art by providing new methods and compositions for safe andeffective tumor vascular targeting, anti-angiogenesis and tumordestruction, which methods and compositions are also surprisinglyeffective in inhibiting viral entry, replication and spread and fortreating viral infections and diseases. The invention is based, in part,on surprising discoveries concerning the expression and role of anionicphospholipids in tumor vasculature and the involvement ofaminophospholipids and anionic phospholipids in viral entry, replicationand spread. The present invention further provides particularlyadvantageous antibodies and immunoconjugates that bind toaminophospholipids and anionic phospholipids, and a new class ofpeptide-based derivatives that bind to phosphatidylethanolamine.

[0017] Overview: In a first overall embodiment, the invention providesnew methods for tumor vascular targeting, tumor imaging and treatmentbased upon the unexpected finding that anionic phospholipids, such asphosphatidylinositol (PI), phosphatidic acid (PA) andphosphatidylglycerol (PG), (as well as phosphatidylserine, PS), areaccessible and stably targetable markers of tumor vasculature. Thisembodiment arose from the unexpected discovery that antibodies againstPA, PI, PG, and other anionic phospholipid components, specificallylocalize to the vasculature of solid tumors.

[0018] Further aspects within this embodiment were developed from theunexpected discovery that naked antibodies against anionicphospholipids, such as PA, PI and PG (as well as PS), specificallyinhibit tumor blood vessel angiogenesis and induce tumor vasculaturedestruction and tumor necrosis in vivo in the absence of conjugation toeffector molecules, such as toxins or coagulants. The invention thusprovides safe and effective methods of vascular targeting,anti-angiogenesis and tumor treatment using single componentantibody-based therapeutics that bind to anionic phospholipids.

[0019] An underlying surprising feature of the invention is thattranslocation of anionic phospholipids to the surface of tumor vascularendothelial cells occurs, at least in a significant part, independentlyof cell damage and apoptotic or other cell-death mechanisms. Anionicphospholipid expression in tumor vasculature is therefore not aconsequence of, or a trigger for, cell death and destruction, but occurson morphologically intact vascular endothelial cells. This means thatanionic phospholipid expression on tumor vasculature is not transient,but rather is stable enough to provide a target for therapeuticintervention.

[0020] Given the finding that anionic phospholipids are stably inducedin tumor vasculature, the invention further provides a range of newmethods and compositions for tumor vasculature imaging and destructionusing immunoconjugates of antibodies against anionic phospholipids.These immunoconjugates comprise antibodies against anionic phospholipidsthat are operatively attached to therapeutic agents, such as toxins andcoagulants, and are useful in the specific delivery of diagnostics andtherapeutics to the surface of tumor vascular endothelial cellmembranes. The therapeutic agents are delivered in intimate contact withthe tumor vascular endothelial cell membrane, allowing either rapidentry into the target cell or rapid association with effector cells,components of the coagulation cascade, and such like

[0021] In a second overall embodiment, the invention provides a numberof preferred antibodies that bind to aminophospholipids and anionicphospholipids (and related immunoconjugates and compositions), whichantibodies have structures and properties that provide advantages overthose known in the art. These so-called “second generation” or improvedantibodies will preferably be used in the anti-angiogenic, anti-cancerand anti-viral and other treatment methods disclosed herein.

[0022] The new classes of antibodies that bind to aminophospholipids andanionic phospholipids provided by the present invention overcome variousdrawbacks in the prior art by providing therapeutic antibodies withoutthe pathogenic properties usually associated with antibodies toaminophospholipids and anionic phospholipids in the art. The inventionwas developed, in part, using new immunization and screening techniquesdeveloped from the inventors' unique observations on phospholipidbehaviour in tumor vascular endothelial cells, and distancing theantibodies generated from anti-phospholipid antibodies associated withdisease. Such antibodies not only have unique properties and improvedsafety, but are equally or more effective than existing antibodies incomparative studies. The compositions and methods of these aspects ofinvention also extend to the use of immunoconjugates and combinations,using the specific category of antibodies provided.

[0023] Prior to the present invention, antibodies that bind toaminophospholipids and anionic phospholipids and have the properties ofthe new antibodies disclosed herein were not known. However, in light ofthe invention disclosed herein, the art is now provided with themethodology for generating new candidate antibodies and with thetechniques to test such antibodies to identify further useful antibodiesfrom the pool of candidates. In light of this invention, therefore, arange of antibodies with advantageous properties and aminophospholipidand anionic phospholipid binding profiles can be made that do not sufferfrom the notable drawbacks and side effects associated with the priorart antibodies. Such antibodies can thus be used in a variety ofembodiments, including in the inhibition of angiogenesis and thetreatment of cancer and viral infections.

[0024] In addition to the new immunization and screening techniquesprovided herein, antibodies that bind to aminophospholipids and anionicphospholipids and have a number of advantageous properties can now beidentified by competition and/or functional assays using the monoclonalantibodies 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 or 3G4. Currently, the 1B12,3B10, 9D2 and 3G4 antibodies are preferred, as these antibodies do notrequire serum for phospholipid binding. The monoclonal antibodies 9D2and 3G4 are more preferred, with monoclonal antibody 3G4 (ATCC 4545)currently being the most preferred. To identify additional antibodiesthat compete with any of the foregoing antibodies, preferably 3G4, thepreferred assays are currently competition assays based upon an ELISA, anumber of which are described herein, and working examples of which aredisclosed.

[0025] In a third overall embodiment, the present invention provides anew class of cell-impermeant peptide-based derivatives that bind to theaminophospholipid, phosphatidylethanolamine (PE). These “PE-bindingpeptide derivatives” comprise at least a first PE-binding peptide,preferably duramycin, which has been modified to substantially preventnon-specific toxicity, preferably by modifying the PE-binding peptide,preferably duramycin, to form a substantially cell impermeant orsubstantially non-pore forming PE-binding construct.

[0026] The generation of a “substantially cell impermeant” or“substantially non-pore forming” PE-binding construct or duramycin ispreferably achieved by attaching the PE-binding peptide or duramycin toat least a first cell impermeant group, preferably a group that preventsclustering of the PE-binding peptide or duramycin. The synthesis of anumber of exemplary duramycin derivatives is described herein. The “cellimpermeant group or groups” may be small molecules, inert carriers, ormay themselves be targeting agents that impart a further targetingfunction to the resultant construct, such as targeting to tumorvasculature. Thus, the PE-binding peptide can be the sole targetingagent linked to an inert carrier, or can be one of two agents that eachimpart a targeting function to the construct. Additionally, PE-bindingpeptides, preferably duramycin, are operatively attached to effectors,such that the PE-binding peptide or duramycin provides the targetingfunction and the attached agent has a substantial therapeutic effectonce delivered to the target cell. Preferred examples are PE-bindingpeptides or duramycin linked to anti-viral agents, such as nucleosides.

[0027] As PE is essentially absent from the surface of normal cellsunder normal conditions, the substantially cell impermeant PE-bindingpeptides of the present invention function to selectively bind to PE atthe surface of aberrant cells or cells associated with disease, such astumor vascular endothelial cells, proliferating and/or virally infectedcells. Upon binding to such aberrant target cells, the PE-bindingconstructs or derivatives inhibit or interrupt PE functions in thosecells, thus resulting in an overall therapeutic benefit, e.g., in thetreatment of tumors and/or viral diseases. The successful use ofsubstantially cell impermeant PE-binding peptides in inhibiting viralentry and spread is disclosed herein. In embodiments where thePE-binding peptides are attached to anti-viral agents, such ascidofovir, enhanced and safer anti-viral treatment is provided.

[0028] In a fourth overall embodiment, the invention further provides animportant new class of compositions and methods for inhibiting viralreplication, infection and spread for use in treating viral infectionsand diseases. These methods are based on the surprising insight thatantibodies and peptides that bind to aminophospholipids and anionicphospholipids, such as PS, PE, PI, PA and PG, particularly PS and PE,would be safe and effective anti-viral agents. Not only has this insightproven to be correct, but the present invention provides data showingthe unexpectedly effective use of antibodies and peptides that bind toaminophospholipids and anionic phospholipids in combating viral spread,meaning that these agents are broadly applicable in the treatment of arange of viral infections and associated diseases.

[0029] These discoveries further encompass new categories ofimmunoconjugates, compositions, kits and methods of use in which anantibody to an aminophospholipid or anionic phospholipid, particularlyPS and PE, is operatively attached to an anti-viral agent. Thesubstantially cell impermeant PE-binding peptide derivatives, such asthe duramycin peptide derivatives, may also be linked to anti-viralagents. Each of these agents thus provide new anti-viral drugs uniquelytargeted to virally infected cells.

[0030] The development of new safe, therapeutic agents effective in thetreatment of aberrant angiogenesis, cancer and viral infections anddiseases is thus a breakthrough in the art.

[0031] Although uniquely effective, the various methods and compositionsof the present invention can also be used to advantage in combinationwith other therapies and agents to provide combined treatment methods,and related compositions, pharmaceuticals and kits of the invention. Ina fifth overall embodiment, therefore, the invention further providesparticular combined compositions, methods and kits, e.g. for cancertreatment, which have been selected and discovered to work surprisinglywell together, as explained in more detail herein.

[0032] Second Generation Antibodies: Certain methods discovered tofunction well in the generation of antibodies with the sought propertiesare described herein in Example IV and embodied in the pending claims.These methods permitted the generation of the advantageous antibodies ofthe invention as exemplified by the monoclonal antibodies 1B9, 1B12,3B10, 2G7, 7C5, 9D2 and 3G4, particularly 3G4 (ATCC 4545).

[0033] The present invention thus provides purified antibodies,antigen-binding fragments and immunoconjugates thereof, which bind to atleast one aminophospholipid or anionic phospholipid, preferably PS, andthat effectively compete with the monoclonal antibody 1B9, 1B12, 3B10,2G7, 7C5, 9D2 or 3G4, preferably with 9D2 or 3G4 (ATCC 4545), and mostpreferably with 3G4, for binding to the aminophospholipid or anionicphospholipid, preferably PS.

[0034] As used throughout the entire application, the terms “a” and “an”are used in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,except in instances wherein an upper limit is thereafter specificallystated. Therefore, an “antibody”, as used herein, means “at least afirst antibody”. The operable limits and parameters of combinations, aswith the amounts of any single agent, will be known to those of ordinaryskill in the art in light of the present disclosure.

[0035] In certain aspects, the antibodies will effectively compete withthe monoclonal antibody 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 or 3G4,preferably with 9D2 or 3G4, and most preferably with 3G4 (ATCC 4545),for binding to an aminophospholipid or anionic phospholipid, preferablyPS, or will have the aminophospholipid or anionic phospholipid bindingprofile of the monoclonal antibody 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 or3G4, preferably of 9D2 or 3G4, and most preferably of 3G4, as set forthin Table 4; and will not be serum dependent, i.e., will not requireserum to bind to the aminophospholipid or anionic phospholipid; not bederived from a patient with a disease, and will not significantlyinhibit coagulation reactions in vitro, cause significant thrombosis invivo or have lupus anticoagulant activities.

[0036] Preferably, such antibodies will also demonstrate an improvementin structural properties or in the range or degree of advantageousfunctional properties in controlled studies in comparison to an antibodyin the literature, such as being IgG, having a higher affinity ordemonstrating enhanced binding to activated endothelial cells, increasedinhibition of endothelial cell proliferation or angiogenesis, improvedtumor blood vessel localization, anticancer and/or anti-viral effects.

[0037] Particular aspects of the invention are therefore based on theinventors' original, surprising generation of antibodies having theforegoing, other disclosed and inherent advantageous properties. Nowthat a panel of preferred antibodies, and a number of particularlypreferred antibodies, have been provided, the present invention furtherencompasses a class of antibodies of defined epitope-specificity,wherein such antibodies, or antigen-binding fragments thereof,effectively compete with the monoclonal antibody 1B9, 1B12, 3B10, 2G7,7C5, 9D2 or 3G4, preferably with 9D2 or 3G4, and most preferably with3G4 (ATCC 4545), for antigen binding, such that they bind to essentiallythe same epitope as the monoclonal antibody 1B9, 1B12, 3B10, 2G7, 7C5,9D2 or 3G4, preferably with 9D2 or 3G4, and most preferably with 3G4(ATCC 4545).

[0038] The invention as claimed is enabled in accordance with thepresent specification and readily available technological references,know-how and starting materials. Nonetheless, on behalf of the presentApplicant, Board of Regents, The University of Texas System, samples ofthe hybridoma cell line producing the 3G4 monoclonal antibody weresubmitted for deposit with the American Type Culture Collection (ATCC),10801 University Blvd., Manassas, Va. 20110-2209, U.S.A. The sampleswere submitted by Avid Bioservices, Inc., 14272 Franklin Avenue, Tustin,Calif. 92780, U.S.A., a subsidiary of the licensee, PeregrinePharmaceuticals, Inc., during the week beginning Jul. 8, 2002, werereceived on July 10 and Jul. 12, 2002, shown to be viable, and givenATCC Accession number PTA 4545 on Jul. 30, 2002.

[0039] This deposit was made under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and the regulations thereof (BudapestTreaty). The hybridoma will be made available by the ATCC under theterms of the Budapest Treaty upon issue of a U.S. patent with pertinentclaims. Availability of the deposited hybridoma is not to be construedas a license to practice the invention in contravention of the rightsgranted under the authority of any government in accordance with itspatent laws.

[0040] In light of the panel of antibodies, the preferred antibodies andthe techniques disclosed herein and known in the art, those of ordinaryskill in the art are now provided with a new class antibodies that bindto aminophospholipids or anionic phospholipids and have advantageousproperties. These antibodies are “like” or “based on” the monoclonalantibodies 1B9, 1B12, 3B10, 2G7, 7C5, 9D2 or 3G4. Preferably, theantibodies of the invention are “9D2-based or 9D2-like antibodies”, andmost preferably, the antibodies of the invention are “3G4-based or3G4-like antibodies”. The following description of “like” antibodies isprovided in terms of the 3G4 antibody (ATCC 4545) for simplicity, but isspecifically incorporated herein by reference as applicable to each ofthe 1B9, 1B12, 3B10, 2G7, 7C5 and 9D2 antibodies.

[0041] A 3G4-like antibody is an antibody, or antigen-binding fragmentthereof, that binds to substantially the same epitope as the monoclonalantibody 3G4 (ATCC 4545) or that binds to at least a firstaminophospholipid or anionic phospholipid, preferably PS, at essentiallythe same epitope as the monoclonal antibody 3G4 (ATCC 4545). Preferably,the antibody, or antigen-binding fragment thereof, will bind to the sameepitope as the monoclonal antibody 3G4 (ATCC 4545).

[0042] The terms “that bind to about, substantially or essentially thesame, or the same, epitope as” the monoclonal antibody 3G4 (ATCC 4545)mean that an antibody “cross-reacts” with the monoclonal antibody 3G4(ATCC 4545). “Cross-reactive antibodies” are those that recognize, bindto or have immunospecificity for substantially or essentially the same,or the same, epitope, epitopic site or common aminophospholipid oranionic phospholipid epitope as the monoclonal antibody 3G4 (ATCC 4545)such that are able to effectively compete with the monoclonal antibody3G4 (ATCC 4545) for binding to at least one aminophospholipid or anionicphospholipid, more than one aminophospholipid or anionic phospholipid orto all aminophospholipid or anionic phospholipids to which themonoclonal antibody 3G4 (ATCC 4545) binds. “3G4-cross-reactiveantibodies” are succinctly termed “3G4-like antibodies” and “3G4-basedantibodies”, and such terms are used interchangeably herein and apply tocompositions, uses and methods.

[0043] The identification of one or more antibodies that bind(s) toabout, substantially, essentially or at the same epitope as themonoclonal antibody 3G4 (ATCC 4545) is a straightforward technicalmatter now that 3G4, with its advantageous properties, has beenprovided. As the identification of cross-reactive antibodies isdetermined in comparison to a reference antibody, it will be understoodthat actually determining the epitope to which the reference antibody(3G4) and the test antibody bind is not in any way required in order toidentify an antibody that binds to the same or substantially the sameepitope as the monoclonal antibody 3G4. However, considerableinformation on the epitope bound by 3G4 is included herein and epitopemapping can be further performed.

[0044] The identification of cross-reactive antibodies can be readilydetermined using any one of variety of immunological screening assays inwhich antibody competition can be assessed. All such assays are routinein the art and are further described herein in detail. Each of U.S. Pat.Nos. 6,342,219, 6,342,221, 6,524,583, and 6,416,758 are specificallyincorporated herein by reference for purposes including even furthersupplementing the present teaching concerning how to make antibodiesthat bind to the same or substantially or essentially the same epitopeas a given antibody, such as 3G4, or that effectively compete with agiven antibody for binding to an antigen.

[0045] For example, where the test antibodies to be examined areobtained from different source animals, or are even of a differentisotype, a simple competition assay may be employed in which the control(3G4) and test antibodies are admixed (or pre-adsorbed) and applied toan aminophospholipid or anionic phospholipid antigen composition,preferably PS. By “aminophospholipid or anionic phospholipid antigencomposition” is meant any composition that contains a 3G4-bindingantigen as described herein, such as described in Table 4. Thus,protocols based upon ELISAs and Western blotting are suitable for use insuch simple competition studies.

[0046] In certain embodiments, one would or pre-mix the controlantibodies (3G4) with varying amounts of the test antibodies (e.g., 1:10or 1:100) for a period of time prior to applying to an antigencomposition. In other embodiments, the control and varying amounts oftest antibodies can simply be admixed during exposure to the antigencomposition. In any event, by using species or isotype secondaryantibodies one will be able to detect only the bound control antibodies,the binding of which will be reduced by the presence of a test antibodythat recognizes substantially the same epitope.

[0047] In conducting an antibody competition study between a controlantibody and any test antibody (irrespective of species or isotype), onemay first label the control (3G4) with a detectable label, such as,e.g., biotin or an enzymatic (or even radioactive) label to enablesubsequent identification. In these cases, one would pre-mix or incubatethe labeled control antibodies with the test antibodies to be examinedat various ratios (e.g., 1:10, 1:100 or 1:1000) and (optionally after asuitable period of time) then assay the reactivity of the labeledcontrol antibodies and compare this with a control value in which nopotentially competing test antibody was included in the incubation.

[0048] The assay may again be any one of a range of immunological assaysbased upon antibody hybridization, and the control antibodies would bedetected by means of detecting their label, e.g., using streptavidin inthe case of biotinylated antibodies or by using a chromogenic substratein connection with an enzymatic label (such as3,3′5,5′-tetramethylbenzidine (TMB) substrate with peroxidase enzyme) orby simply detecting a radioactive label. An antibody that binds to thesame epitope as the control antibodies will be able to effectivelycompete for binding and thus will significantly reduce control antibodybinding, as evidenced by a reduction in bound label.

[0049] The reactivity of the (labeled) control antibodies in the absenceof a completely irrelevant antibody would be the control high value. Thecontrol low value would be obtained by incubating the labeled (3G4)antibodies with unlabelled antibodies of exactly the same type (3G4),when competition would occur and reduce binding of the labeledantibodies. In a test assay, a significant reduction in labeled antibodyreactivity in the presence of a test antibody is indicative of a testantibody that recognizes the same epitope, i.e., one that “cross-reacts”with the labeled (3G4) antibody.

[0050] A significant reduction is a “reproducible”, i.e., consistentlyobserved, reduction in binding. A “significant reduction” in terms ofthe present application is defined as a reproducible reduction (in 3G4binding to one or more aminophospholipid or anionic phospholipids,preferably PS, in an ELISA) of at least about 70%, about 75% or about80% at any ratio between about 1:10 and about 1:1000. Antibodies witheven more stringent cross-blocking activities will exhibit areproducible reduction (in 3G4 binding to one or more aminophospholipidor anionic phospholipids, preferably PS, in an ELISA or other suitableassay) of at least about 82%, about 85%, about 88%, about 90%, about 92%or about 95% or so at any ratio between about 1:10 and about 1:1000.Complete or near-complete cross-blocking, such as exhibiting areproducible reduction in 3G4 binding to one or more aminophospholipidor anionic phospholipids of about 97% or about 96% or so, although by nomeans required to practice the invention, is certainly not excluded.

[0051] As to the second generation antibodies overall, the competitionmay be measured in reference to an antibody that at least binds tophosphatidylserine, wherein the second generation antibody effectivelycompetes for binding to phosphatidylserine; in reference to an antibodythat at least binds to phosphatidic acid, wherein the second generationantibody effectively competes for binding to phosphatidic acid; inreference to an antibody that at least binds to phosphatidylinositol,wherein the second generation antibody effectively competes for bindingto phosphatidylinositol; in reference to an antibody that at least bindsto phosphatidylglycerol, wherein the second generation antibodyeffectively competes for binding to phosphatidylglycerol; in referenceto an antibody that at least binds to cardiolipin, wherein the secondgeneration antibody effectively competes for binding to cardiolipin; andoptionally in reference to an antibody that at least binds tophosphatidylethanolamine, wherein the second generation antibodyeffectively competes for binding to phosphatidylethanolamine.

[0052] In certain embodiments, the second generation antibodies may bemeasured in reference to an antibody that binds to at least a first andsecond aminophospholipid or anionic phospholipid, and wherein the secondgeneration antibody effectively competes for binding to the first andsecond aminophospholipid or anionic phospholipid; in reference to anantibody that binds to at least a first, second and thirdaminophospholipid or anionic phospholipid, and wherein the secondgeneration antibody effectively competes for binding to the first,second and third aminophospholipid or anionic phospholipid; in referenceto an antibody that binds to at least a first, second, third and fourthaminophospholipid or anionic phospholipid, and wherein the secondgeneration antibody effectively competes for binding to the first,second, third and fourth aminophospholipid or anionic phospholipid; orin reference to an antibody that binds to at least a first, second,third, fourth and fifth aminophospholipid or anionic phospholipid, andwherein the second generation antibody effectively competes for bindingto the first, second, third, fourth and fifth aminophospholipid oranionic phospholipid.

[0053] In further embodiments, a second generation antibody maycharacterized as an antibody that exhibits significant binding to atleast one aminophospholipid or anionic phospholipid, no detectablebinding to a choline-containing neutral phospholipid and thateffectively competes with a monoclonal antibody of the invention,preferably 3G4 (ATCC 4545).

[0054] In particular embodiments, the antibody exhibits significantbinding to the anionic phospholipids PS, PA, PI, PG and CL; has aphospholipid binding profile of PS=PA=PI=PG>CL>>PE, wherein > indicatesat least 2-fold difference in binding and >> indicates at least 10-folddifference in binding to such phospholipids; exhibits no detectablebinding to phosphatidylcholine or sphingomyelin; and effectivelycompetes with the monoclonal antibody 3G4 (ATCC 4545) for binding toeach of the anionic phospholipids PS, PA, PI PG and CL.

[0055] Preferably, the second generation antibodies will have theforegoing characteristics and also exhibits significant binding to atleast one anionic phospholipid present at the cell surface of activated,dividing, injured, apoptotic or virally infected cells. More preferably,the antibody also significantly inhibits the proliferation of dividingendothelial cells without significantly altering quiescent cells, andmore preferably, has no significant lupus anticoagulant activities.

[0056] Functionally, the second generation antibodies will preferablysuppresses angiogenesis, have an anti-tumor effect and an anti-viraleffect, preferably in vivo, and more preferably, will do so withoutcausing significant thrombotic complications in animals or patients.Thus, the preferred antibodies possess the combined properties of ananti-angiogenic, anti-tumor vascular, anti-tumor and anti-viral agent.

[0057] The invention is exemplified by monoclonal antibody 3G4, producedby hybridoma ATCC 4545, or an antigen-binding fragment of such amonoclonal antibody. A hybridoma that produces a monoclonal antibodythat binds to substantially the same epitope as the monoclonal antibody3G4 (ATCC 4545) is another aspect of the invention.

[0058] The invention further provides antibodies that bind tosubstantially the same epitope as the monoclonal antibody 3G4 (ATCC4545), prepared by a process comprising immunizing an animal with acomposition comprising at least a first immunogenic aminophospholipid oranionic phospholipid, including a composition comprising activatedendothelial cells, and selecting from the immunized animal an antibodythat substantially cross-reacts with the monoclonal antibody 3G4 (ATCC4545); and antibodies that bind to substantially the same epitope as themonoclonal antibody 3G4 (ATCC 4545), prepared by a process comprisingimmunizing an animal with a composition comprising at least a firstimmunogenic aminophospholipid or anionic phospholipid, including acomposition comprising activated endothelial cells, and selecting acompeting antibody from the immunized animal by identifying an antibodythat substantially reduces the binding of the 3G4 (ATCC 4545) antibodyto at least a first aminophospholipid or anionic phospholipid,preferably PS.

[0059] In the following descriptions of the compositions,immunoconjugates, pharmaceuticals, combinations, cocktails, kits, firstand second medical uses and all methods in accordance with thisinvention, the terms “antibody” and “immunoconjugate”, or anantigen-binding region thereof, unless otherwise specifically stated ormade clear from the scientific terminology, refer to a range ofanti-aminophospholipid or anti-anionic phospholipid antibodies as wellas to specific 3G4-cross-reactive antibodies.

[0060] The terms “antibody” and “immunoglobulin”, as used herein, referbroadly to any immunological binding agent, including polyclonal andmonoclonal antibodies. Depending on the type of constant domain in theheavy chains, antibodies are assigned to one of five major classes: IgA,IgD, IgE, IgG, and IgM. Several of these are further divided intosubclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like.The heavy-chain constant domains that correspond to the differenceclasses of immunoglobulins are termed α, β, ε, γ and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

[0061] Generally, where antibodies rather than antigen binding regionsare used in the invention, IgG and/or IgM are preferred because they arethe most common antibodies in the physiological situation and becausethey are most easily made in a laboratory setting. The “light chains” ofmammalian antibodies are assigned to one of two clearly distinct types:kappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains. There is essentially no preference to the use of κ orλ light chains in the antibodies of the present invention.

[0062] The use of monoclonal antibodies (MAbs) or derivatives thereof ismuch preferred. MAbs are recognized to have certain advantages, e.g.,reproducibility and large-scale production, which makes them suitablefor clinical treatment. The invention thus provides monoclonalantibodies of the murine, human, monkey, rat, hamster, rabbit and evenfrog or chicken origin. Murine, human or humanized monoclonal antibodieswill generally be preferred.

[0063] As will be understood by those in the art, the immunologicalbinding reagents encompassed by the term “antibody” extend to allantibodies from all species, and antigen binding fragments thereof,including dimeric, trimeric and multimeric antibodies; bispecificantibodies; chimeric antibodies; human and humanized antibodies;recombinant, engineered and camelized (camelised) antibodies, andfragments thereof.

[0064] The term “antibody” is thus used to refer to any antibody-likemolecule that has an antigen binding region, and this term includesantibody fragments such as Fab′, Fab, F(ab′)₂, single domain antibodies(DABs), Fv, scFv (single chain Fv), linear antibodies, diabodies,camelized antibodies and the like. The techniques for preparing andusing various antibody-based constructs and fragments are well known inthe art (see Kabat et al., 1991, specifically incorporated herein byreference). Diabodies, in particular, are further described in EP404,097 and WO 93/11161, each specifically incorporated herein byreference; whereas linear antibodies are further described in Zapata etal. (1995), specifically incorporated herein by reference.

[0065] The antibodies of the invention include those that bind tophosphatidylserine and comprises at least one CDR of an antibodyprovided herein, preferably the 9D2 or 3G4 (ATCC 4545) antibody. Forexample, the invention provides antibodies that bind tophosphatidylserine and comprise at least one CDR from the monoclonalantibody 3G4 produced by the hybridoma deposited as ATCC PTA 4545; or atleast one CDR that has the amino acid sequence of SEQ ID NO:2 or SEQ IDNO:4, or a variant or mutagenized form of the amino acid sequence of SEQID NO:2 or SEQ ID NO:4, wherein such a variant or mutagenized formmaintains binding to phosphatidylserine.

[0066] Certain antibodies thus comprise at least one CDR from thevariable regions of each of the heavy and light chains of monoclonalantibody 3G4 (ATCC 4545), at least one CDR1-3 of the monoclonal antibody3G4 (ATCC 4545), or CDR1-3 of the variable regions of each of the heavyand light chains of monoclonal antibody 3G4 (ATCC PTA 4545). Otherantibodies comprise at least a first variable region that includes anamino acid sequence region having the amino acid sequence of SEQ ID NO:2or SEQ ID NO:4, as exemplified by variable regions that include an aminoacid sequence region encoded by the nucleic acid sequences of SEQ IDNO:1 or SEQ ID NO:3. Such sequences are the sequences of Vh and Vκ ofthe 3G4 ScFv encompassing CDR1-3 (complementarity determining regions)of the variable regions of the heavy and light chains.

[0067] In certain embodiments, second generation antibodies are providedthat have enhanced or superior properties in comparison to an originalanti-aminophospholipid or anti-anionic phospholipid antibody, such as3G4 (ATCC 4545). These are exemplified by antibodies that comprise atleast one CDR that has a variant or mutagenized form of the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:4, wherein such a variant ormutagenized form maintains binding to phosphatidylserine.

[0068] The anti-aminophospholipid or anti-anionic phospholipidantibodies thus include those that comprises at least a first variableregion that includes an amino acid sequence region of at least about75%, more preferably, at least about 80%, more preferably, at leastabout 85%, more preferably, at least about 90% and most preferably, atleast about 95% or so amino acid sequence identity to the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:4; wherein saidanti-aminophospholipid or anti-anionic phospholipid antibody at leastsubstantially maintains the biological properties of theanti-aminophospholipid or anti-anionic phospholipid antibodies of thepresent invention, as exemplified by the 3G4 antibody.

[0069] Identity or homology with respect to these and otheranti-aminophospholipid or anti-anionic phospholipid antibody sequencesof the present invention is defined herein as the percentage of aminoacid residues in a candidate sequence that are identical to thesequences of SEQ ID NO:2 or SEQ ID NO:4, or to the sequence of anotheranti-aminophospholipid or anti-anionic phospholipid antibody of theinvention, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Themaintenance of substantially the same, or even more effective biologicalproperties of the anti-aminophospholipid or anti-anionic phospholipidantibody used for the sequence comparison is particularly important.Such comparisons are easily conducted, e.g., using one or more of thevarious assays described in detail herein.

[0070] In further embodiments, the antibodies employed will be“humanized”, part-human or human antibodies. “Humanized” antibodies aregenerally chimeric monoclonal antibodies from mouse, rat, or othernon-human species, bearing human constant and/or variable region domains(“part-human chimeric antibodies”). Various humanized monoclonalantibodies for use in the present invention will be chimeric antibodieswherein at least a first antigen binding region, or complementaritydetermining region (CDR), of a mouse, rat or other non-human monoclonalantibody is operatively attached to, or “grafted” onto, a human antibodyconstant region or “framework”.

[0071] “Humanized” monoclonal antibodies for use herein may also bemonoclonal antibodies from non-human species wherein one or moreselected amino acids have been exchanged for amino acids more commonlyobserved in human antibodies. This can be readily achieved through theuse of routine recombinant technology, particularly site-specificmutagenesis.

[0072] Entirely human, rather than “humanized”, antibodies may also beprepared and used in the present invention. Such human antibodies may beobtained from healthy subjects by simply obtaining a population of mixedperipheral blood lymphocytes from a human subject, includingantigen-presenting and antibody-producing cells, and stimulating thecell population in vitro by admixing with an immunogenically effectiveamount of an aminophospholipid or anionic phospholipid sample. The humananti-aminophospholipid or anti-anionic phospholipid antibody-producingcells, once obtained, are used in hybridoma and/or recombinant antibodyproduction.

[0073] Further techniques for human monoclonal antibody productioninclude immunizing a transgenic animal, preferably a transgenic mouse,which comprises a human antibody library with an immunogenicallyeffective amount of an aminophospholipid or anionic phospholipid sample.This also generates human anti-aminophospholipid or anti-anionicphospholipid antibody-producing cells for further manipulation inhybridoma and/or recombinant antibody production, with the advantagethat spleen cells, rather than peripheral blood cells, can be readilyobtained from the transgenic animal or mouse.

[0074] Antibodies in accordance with the invention may be readilyprepared by selecting an antibody that substantially cross-reacts orcompetes with the monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545).Suitable preparative processes and methods comprise:

[0075] (a) preparing candidate antibody-producing cells; and

[0076] (b) selecting from the candidate antibody-producing cells anantibody that substantially cross-reacts or competes with the monoclonalantibody 9D2 or 3G4 (ATCC PTA 4545).

[0077] One process of preparing suitable antibody-producing cells andobtaining antibodies therefrom may be conduced in situ in a givenpatient. That is, simply providing an immunogenically effective amountof an immunogenic aminophospholipid or anionic phospholipid sample to apatient will result in appropriate antibody generation. Thus, theantibody is still “obtained” from the antibody-producing cell, but itdoes not have to be isolated away from a host and subsequently providedto a patient, being able to spontaneously localize to the tumorvasculature and exert its biological anti-tumor effects. However, suchembodiments are not currently preferred.

[0078] Suitable antibody-producing cells may also be obtained, andantibodies subsequently isolated and/or purified, by stimulatingperipheral blood lymphocytes with aminophospholipid or anionicphospholipid in vitro.

[0079] Other methods comprise administering to an animal an immunizingcomposition comprising at least a first immunogenic aminophospholipid oranionic phospholipid component and selecting from the immunized animalan antibody that substantially cross-reacts or competes with themonoclonal antibody 9D2 or 3G4 (ATCC PTA 4545). These methods generallycomprise:

[0080] (a) immunizing an animal by administering to the animal at leastone dose, and optionally more than one dose, of a composition comprisingan immunogenically effective amount of an immunogenic aminophospholipidor anionic phospholipid; and

[0081] (b) obtaining a suitable antibody-producing cell from theimmunized animal, such as an antibody-producing cell that produces anantibody that substantially cross-reacts or competes with the monoclonalantibody 9D2 or 3G4 (ATCC PTA 4545).

[0082] A preferred “composition comprising an immunogenically effectiveamount of an immunogenic aminophospholipid or anionic phospholipid”, asused herein, is a composition comprising activated endothelial cells.“Activated endothelial cells” are preferably prepared by placingendothelial cells under at least a first condition, or in contact withat least a first factor, which activates the endothelial cells, and/ormimics a tumor environment, for a time effective to substantiallymaintain cell viability and stimulate expression of at least one anionicphospholipid at the surface of the endothelial cells.

[0083] Examples “conditions” effective to prepare activated endothelialcells are hypoxic and/or acidic environments. Examples of “factors”effective to prepare activated endothelial cells are effectiveconcentrations of H₂O₂, thrombin, inflammatory cytokine(s), such asIL-1α, IL-1β, interferon or TNFα, and generally, combinations ofconditions and/or factors that mimic a tumor environment.

[0084] Irrespective of the nature of the immunization process, or thetype of immunized animal, suitable antibody-producing cells are obtainedfrom the immunized animal and, preferably, further manipulated by thehand of man. “An immunized animal”, as used herein, is a non-humananimal, unless otherwise expressly stated. Although anyantibody-producing cell may be used, most preferably, spleen cells areobtained as the source of the antibody-producing cells. Theantibody-producing cells may be used in a preparative process thatcomprises:

[0085] (a) fusing a suitable anti-aminophospholipid or anti-anionicphospholipid antibody-producing cell with an immortal cell to prepare ahybridoma that produces a monoclonal antibody in accordance with thepresent invention; and

[0086] (b) obtaining a suitable anti-aminophospholipid or anti-anionicphospholipid antibody in accordance with the invention from thehybridoma.

[0087] “Suitable” anti-aminophospholipid or anti-anionic phospholipidantibody-producing cells, hybridomas and antibodies are those thatproduce, or exist as, anti-aminophospholipid or anti-anionicphospholipid antibodies, preferably antibodies that substantiallycross-react or compete with the monoclonal antibody 9D2 or 3G4 (ATCC PTA4545).

[0088] Hybridoma-based monoclonal antibody preparative methods thusinclude those that comprise:

[0089] (a) immunizing an animal by administering to the animal at leastone dose, and optionally more than one dose, of a composition comprisingan immunogenically effective amount of an immunogenic aminophospholipidor anionic phospholipid, preferably a composition comprising activatedendothelial cells;

[0090] (b) preparing a collection of monoclonal antibody-producinghybridomas from the immunized animal;

[0091] (c) selecting from the collection at least a first hybridoma thatproduces at least a first anti-aminophospholipid or anti-anionicphospholipid monoclonal antibody in accordance with the invention,optionally an anti-aminophospholipid or anti-anionic phospholipidantibody that substantially cross-reacts or competes with the monoclonalantibody 9D2 or 3G4 (ATCC PTA 4545); and

[0092] (d) culturing the at least a first antibody-producing hybridomato provide the at least a first anti-aminophospholipid or anti-anionicphospholipid monoclonal antibody; and preferably

[0093] (e) obtaining the at least a first anti-aminophospholipid oranti-anionic phospholipid monoclonal antibody from the cultured at leasta first hybridoma.

[0094] In identifying an anti-aminophospholipid or anti-anionicphospholipid antibody that substantially cross-reacts with themonoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), the selecting step maycomprise:

[0095] (a) contacting an aminophospholipid or anionic phospholipidsample, preferably a PS sample, with effective amounts of the monoclonalantibody 9D2 or 3G4 (ATCC PTA 4545) and a candidate antibody; and

[0096] (b) determining the ability of the candidate antibody tosubstantially reduce the binding of the 9D2 or 3G4 antibody to theaminophospholipid or anionic phospholipid, preferably PS, sample;wherein the ability of a candidate antibody to substantially reduce thebinding of the 9D2 or 3G4 antibody to the aminophospholipid or anionicphospholipid, preferably PS sample is indicative of ananti-aminophospholipid or anti-anionic phospholipid antibody that bindsto substantially the same epitope as the monoclonal antibody 9D2 or 3G4(ATCC PTA 4545).

[0097] The selecting step may further comprise:

[0098] (a) contacting a first aminophospholipid or anionic phospholipidsample, preferably PS, with an effective binding amount of themonoclonal antibody 9D2 or 3G4 (ATCC PTA 4545) and determining theamount of 9D2 or 3G4 that binds to he aminophospholipid or anionicphospholipid, preferably PS;

[0099] (b) contacting a second aminophospholipid or anionic phospholipidsample, preferably PS, with an effective binding amount of themonoclonal antibody 9D2 or 3G4 (ATCC PTA 4545) in combination with aneffective competing amount of a candidate antibody and determining theamount of 9D2 or 3G4 that binds to the aminophospholipid or anionicphospholipid, preferably PS, in the presence of the candidate antibody;and

[0100] (c) identifying an anti-aminophospholipid or anti-anionicphospholipid antibody that binds to substantially the same epitope asthe monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545) by selecting acandidate antibody that reduces the amount of 9D2 or 3G4 that binds tothe aminophospholipid or anionic phospholipid, preferably PS, preferablyby at least about 80%.

[0101] All selection criteria, as used herein, are preferably conductedin the absence of serum, to avoid the drawbacks with generatingantibodies that could mimic the pathological antibodies of patients,which bind to aminophospholipids or anionic phospholipids in conjunctionwith proteins.

[0102] As non-human animals are used for immunization, the monoclonalantibodies obtained from such a hybridoma will often have a non-humanmake up. Such antibodies may be optionally subjected to a humanizationprocess, grafting or mutation, as known to those of skill in the art andfurther disclosed herein. Alternatively, transgenic animals, such asmice, may be used that comprise a human antibody gene library.Immunization of such animals will therefore directly result in thegeneration of suitable human antibodies.

[0103] After the production of a suitable antibody-producing cell, mostpreferably a hybridoma, whether producing human or non-human antibodies,the monoclonal antibody-encoding nucleic acids may be cloned to preparea “recombinant” monoclonal antibody. Any recombinant cloning techniquemay be utilized, including the use of PCR™ to prime the synthesis of theantibody-encoding nucleic acid sequences. Therefore, yet furtherappropriate monoclonal antibody preparative methods include those thatcomprise using the antibody-producing cells as follows:

[0104] (a) obtaining at least a first suitable anti-aminophospholipid oranti-anionic phospholipid antibody-encoding nucleic acid molecule orsegment from a suitable anti-aminophospholipid or anti-anionicphospholipid antibody-producing cell, preferably a hybridoma; and

[0105] (b) expressing the nucleic acid molecule or segment in arecombinant host cell to obtain a recombinant anti-aminophospholipid oranti-anionic phospholipid monoclonal antibody in accordance with thepresent invention.

[0106] However, other powerful recombinant techniques are available thatare ideally suited to the preparation of recombinant monoclonalantibodies. Such recombinant techniques include the phagemidlibrary-based monoclonal antibody preparative methods comprising:

[0107] (a) immunizing an animal by administering to the animal at leastone dose, and optionally more than one dose, of a composition comprisingan immunogenically effective amount of an immunogenic aminophospholipidor anionic phospholipid, preferably a composition comprising activatedendothelial cells;

[0108] (b) preparing a combinatorial immunoglobulin phagemid libraryexpressing RNA isolated from the antibody-producing cells, preferablyfrom the spleen, of the immunized animal;

[0109] (c) selecting from the phagemid library at least a first clonethat expresses at least a first anti-aminophospholipid or anti-anionicphospholipid antibody, optionally one that substantially cross-reacts orcompetes with the monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545);

[0110] (d) obtaining anti-aminophospholipid or anti-anionic phospholipidantibody-encoding nucleic acids from the at least a first selected cloneand expressing the nucleic acids in a recombinant host cell to providethe at least a first anti-aminophospholipid or anti-anionic phospholipidantibody; and preferably

[0111] (e) obtaining the at least a first anti-aminophospholipid oranti-anionic phospholipid antibody expressed by the nucleic acidsobtained from the at least a first selected clone.

[0112] Again, in such phagemid library-based techniques, transgenicanimals bearing human antibody gene libraries may be employed, thusyielding recombinant human monoclonal antibodies.

[0113] Irrespective of the manner of preparation of a firstanti-aminophospholipid or anti-anionic phospholipid antibody nucleicacid segment, further suitable antibody nucleic acid segments may bereadily prepared by standard molecular biological techniques. In orderto confirm that any variant, mutant or second generationanti-aminophospholipid or anti-anionic phospholipid antibody nucleicacid segment is suitable for use in the present invention, the nucleicacid segment will be tested to confirm expression of ananti-aminophospholipid or anti-anionic phospholipid antibody inaccordance with the present invention. Preferably, the variant, mutantor second generation nucleic acid segment will also be tested to confirmhybridization under standard, more preferably, standard stringenthybridization conditions. Exemplary suitable hybridization conditionsinclude hybridization in about 7% sodium dodecyl sulfate (SDS), about0.5 M NaPO₄, about 1 mM EDTA at about 50° C.; and washing with about 1%SDS at about 42° C.

[0114] As a variety of recombinant monoclonal antibodies, whether humanor non-human in origin, may be readily prepared, any of the treatmentmethods of the invention may be executed by providing to the animal orpatient at least a first nucleic acid segment that expresses abiologically effective amount of at least a first anti-aminophospholipidor anti-anionic phospholipid antibody in the patient. The “nucleic acidsegment that expresses an anti-aminophospholipid or anti-anionicphospholipid, 3G4-like or 3G4-based antibody” will generally be in theform of at least an expression construct, and may be in the form of anexpression construct comprised within a virus or within a recombinanthost cell. Preferred gene therapy vectors of the present invention willgenerally be viral vectors, such as comprised within a recombinantretrovirus, herpes simplex virus (HSV), adenovirus, adeno-associatedvirus (AAV), cytomegalovirus (CMV), and the like.

[0115] Cell Impermeant Duramycin Derivatives: The invention furtherprovides substantially cell impermeant phosphatidylethanolamine(PE)-binding peptide constructs and derivatives, which comprise at leasta first PE-binding peptide that has been modified to form asubstantially cell impermeant PE-binding construct.

[0116] Preferably, the invention provides pharmaceutical compositionscomprising, in a pharmaceutically acceptable carrier, a biologically ortherapeutically effective amount of at least a first substantially cellimpermeant PE-binding construct, which comprises at least a firstPE-binding peptide that has been modified to form a substantially cellimpermeant PE-binding construct. Thus, the substantially cell impermeantPE-binding constructs are constructs for pharmaceutical, pharmacologicaland therapeutic, i.e., medical uses, preferably for use in treatingviral infections. In certain embodiments, the invention provides asubstantially cell impermeant PE-binding construct other than cinnamycinlinked to biotin.

[0117] Most preferably, the substantially cell impermeant PE-bindingpeptide derivatives of the invention are substantially cell impermeantduramycin peptide derivatives and pharmaceutical compositions thereof.The duramycin peptide is typically modified to form a substantially cellimpermeant duramycin derivative by operative attachment to at least afirst substantially cell impermeant group. Operative attachment of asubstantially cell impermeant group may be via the lysine residue atamino acid position 2 in SEQ ID NO:9.

[0118] The substantially cell impermeant group may have a positive ornegative charge at physiological pH or may be polar at physiological pH.Exemplary groups include sulfate, sulfonate, phosphate, carboxyl,phenolic, quaternary ammonium ion and amine groups. A pharmaceuticalcomposition comprising duramycin linked to biotin is a particularexample within the invention.

[0119] Substantially cell impermeant duramycins may also be operativelyattached to a sugar, oligo- or polysaccharide, amino acid, peptide,polypeptide, protein or a polyalcohol group. Certain cell impermeantduramycins are those operatively attached to a carrier protein or “aninert carrier protein”, such as neutravidin, streptavidin, albumin or animmunoglobulin carrier protein (an inert immunoglobulin carrierprotein), of which duramycin attached to human IgG (HIgG) isparticularly preferred. Other examples of cell impermeant duramycins arethose linked to targeting agents, preferably wherein the targeting agentis a protein, antibody, or antigen binding region thereof, that binds toa component of a tumor cell, tumor vasculature or tumor stroma or to avirally-infected cell. Examples of targeting agents that bind to acomponent of a tumor cell, tumor vasculature or tumor stroma are taughtin U.S. Pat. Nos. 6,093,399, 6,004,555, 5,877,289, and 6,036,955, eachspecifically incorporated herein by reference.

[0120] Conjugates, Compositions and Kits: Unless otherwise specificallystated or made clear in scientific terms, the terms “antibody andfragment thereof”, as used herein, therefore mean an “unconjugated ornaked” antibody or fragment, which is not attached to another agent,particularly a therapeutic or diagnostic agent. These definitions do notexclude modifications of the antibody, such as, by way of example,modifications to improve the biological half life, affinity, avidity orother properties of the antibody, or combinations of the antibody withother effectors.

[0121] Similarly, the terms PE-binding peptide and duramycin“derivative”, as used herein, mean PE-binding and duramycin peptidesthat are not specifically attached to a selected therapeutic agent,particularly an anti-viral agent. Naturally, as the preferred PE-bindingpeptide and duramycin “derivatives” of the invention are alreadyattached to at least a first substantially cell impermeant group, thisdefinition refers to the lack of an attached agent “selected” for atherapeutic effect, particularly an anti-viral effect.

[0122] The invention further provides a range of antibody(immunoconjugate) and peptide conjugates. The immunoconjugates of theinvention comprise an anti-aminophospholipid or anti-anionicphospholipid antibody, preferably one that binds to substantially thesame epitope as, or competes with, the monoclonal antibody 9D2 or 3G4(ATCC PTA 4545), operatively attached to at least a first biological,diagnostic or therapeutic agent. Any of the range of antibodiesdescribed above may be used in such an immunoconjugate.

[0123] The “biological agent” need not directly be a therapeutic ordiagnostic agent. For example, as the invention can be used inconnection with prodrugs, including ADEPT embodiments, the biologicalagent may be an agent, preferably an enzyme, which cleaves asubstantially inactive prodrug to release a substantially active drug.Such agents and enzymes are described below in relation to the prodrugand ADEPT method embodiments.

[0124] As to “diagnostic agents”, preferred diagnostic agents forattachment are in vivo diagnostic agents. Such diagnosticimmunoconjugates may be used in imaging pre-apoptotic and apoptoticcells in a range of diseases, in combined tumor imaging and treatment,and in methods of using the invention as a surrogate marker to monitorchemotherapy.

[0125] Suitable detectable labels include an X-ray detectable compound,such as bismuth (III), gold (III), lanthanum (III) or lead (II); aradioactive ion, such as copper⁶⁷, gallium⁶⁷, gallium⁶⁸, indium¹¹¹,indium¹¹³, iodine¹²³, iodine¹²⁵, iodine¹³¹, mercury¹⁹⁷, mercury²⁰³,rhenium¹⁸⁶, rhenium¹⁸⁸, rubidium⁹⁷, rubidium¹⁰³, technetium^(99m) oryttrium⁹⁰; a nuclear magnetic spin-resonance isotope, such as cobalt(II), copper (II), chromium (III), dysprosium (III), erbium (III),gadolinium (III), holmium (III), iron (II), iron (III), manganese (II),neodymium (III), nickel (II), samarium (III), terbium (III), vanadium(II) or ytterbium (III); or rhodamine or fluorescein.

[0126] Regarding “therapeutic agents”, certain preferred therapeuticagents are cytotoxic, cytostatic or anti-cancer agents. The antibodiesof the invention, preferably 9D2- or 3G4-like antibodies, may thereforebe linked to at least a first radiotherapeutic, chemotherapeutic,anti-cellular, cytotoxic, anti-angiogenic or apoptosis-inducing agent orto an anti-tubulin drug or cytokine.

[0127] Currently preferred agents are the cytotoxic agent, gelonin;cytokines, such as TNFα, IL-12 and LEC (liver-expressed chemokine);anti-cancer agents with anti-angiogenic effects, as in Table E;anti-cancer agents that induce apoptosis, as in Table F; andanti-tubulin drugs from the combretastatin family.

[0128] For attachment to at least a first biological, diagnostic,cytotoxic, cytostatic or anticancer agent, antibodies that bind tosubstantially the same epitope as, i.e., compete with, the monoclonalantibody 9D2 or 3G4 (ATCC PTA 4545) are particularly preferred. Giventhe surprising connection between the antibodies and peptides of theinvention and viral infections, the present invention further provides arange of new therapeutic conjugates for use in treating viralinfections. Other preferred therapeutic agents for attachment toantibodies are therefore anti-viral agents or drugs. The anti-viralimmunoconjugates of the invention comprise an antibody that binds to atleast a first aminophospholipid or anionic phospholipid, preferably toan aminophospholipid, and most preferably to PS or PE, operativelyattached to at least a first anti-viral agent or drug.

[0129] The peptide conjugates of the invention comprise a substantiallycell impermeant PE-binding peptide, preferably a duramycin peptide,operatively attached to at least a first anti-viral agent or drug. Suchpeptide conjugates are herein termed “PE-binding peptide anti-viralconjugates” or succinctly, “anti-viral peptide conjugates”. Any of therange of PE-binding peptides described above may be used in such aconjugate, with duramycin being particularly preferred.

[0130] Virtually any one or more “anti-viral agents or drugs” may beattached to an antibody that binds to at least a first aminophospholipidor anionic phospholipid, preferably to an aminophospholipid, and mostpreferably to PS or PE; or to a PE-binding peptide, preferably aduramycin peptide. Anti-retroviral drugs may be used, for example,nucleoside reverse transcriptase (RT) inhibitors (NTRIs), non-nucleosideRT inhibitors and protease inhibitors. Other suitable anti-viral agentsfor attachment to the antibodies and peptides of the invention includethose set forth in Table G, particularly AZT or cidofovir.

[0131] For antibody- and peptide-based conjugates, the term “conjugate”is generally used to define the operative association of the antibody orpeptide with another effective agent and is not intended to refer solelyto any type of operative association, and is particularly not limited tochemical “conjugation”. Recombinant fusion proteins are particularlycontemplated. So long as the antibody or peptide is able to bind to thetarget aminophospholipid or anionic phospholipid and the attached agentfunctions sufficiently as intended, particularly when delivered to thetarget site, any mode of attachment will be suitable.

[0132] The invention further provides compositions comprising at least afirst purified anti-aminophospholipid or anti-anionic phospholipidantibody, or antigen-binding fragment or immunoconjugate thereof,optionally one that binds to essentially the same epitope as themonoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), or a substantially cellimpermeant PE-binding peptide derivative, preferably a substantiallycell impermeant duramycin derivative, or an anti-viral conjugatethereof. The compositions preferably comprise a biologically effectiveamount of any such agent, such as an amount effective to bind a targetantigen, inhibit proliferation, viral replication or such like.

[0133] The compositions of the invention are preferably pharmaceuticallyacceptable compositions, particularly those for the substantially cellimpermeant PE-binding peptide derivatives, preferably substantially cellimpermeant duramycin derivatives. The pharmaceutical compositionsinclude those formulated for parenteral administration, such as forintravenous administration, or for administration as a liposome or as anaerosol. The aerosol formulations are particularly suitable for treatingviral infections. Pharmaceutical compositions preferably comprise abiologically or therapeutically effective amount of any such agent, suchas an amount effective for treating a disease or disorder, particularlyangiogenesis, cancer or a viral infection.

[0134] Aspects of the invention further include compositions,pharmaceutical compositions, combinations, mixtures, medicaments and/ormedicinal cocktails of agents, comprising at least a first purifiedanti-aminophospholipid or anti-anionic phospholipid antibody, orantigen-binding fragment or immunoconjugate thereof, optionally one thatbinds to essentially the same epitope as the monoclonal antibody 9D2 or3G4 (ATCC PTA 4545), or a substantially cell impermeant PE-bindingpeptide derivative, preferably a substantially cell impermeant duramycinderivative, or an anti-viral conjugate thereof, in combination with abiologically or therapeutically effective amount of at least a secondbiological agent. All such combinations preferably comprise combinedbiologically or therapeutically effective amounts, such as combinedamounts effective to inhibit proliferation or viral replication, or totreat a disease such as an angiogenic disease, cancer or a viralinfection.

[0135] In the compositions, the “at least a second biological agent”will often be a diagnostic or therapeutic agent, but it need not be. Forexample, the second biological agent may be a component of apharmaceutical composition such as a dispersion agent or an absorptiondelaying agent. Other biological agents, such as agents for makingantibodies and prodrugs for use in prodrug and ADEPT methods, anddiagnostic agents, are preferably maintained in combination, butseparately, from the first composition of the invention and aretherefore discussed below in reference to the kits of the invention. “Incombination, but separately” means in close confinement together, butnot part of the same composition, such as not part of the same solutionor pharmaceutical composition.

[0136] As to the “at least a second therapeutic agent”, the term“second” is in reference to the anti-aminophospholipid or anti-anionicphospholipid antibody, fragment or immunoconjugate, or substantiallycell impermeant PE-binding peptide, duramycin derivative or anti-viralconjugate thereof, being the “first” therapeutic agent.

[0137] Where the invention is intended for use in cancer treatment, theat least a second therapeutic agent will preferably be “at least asecond, distinct anti-cancer agent”. The second, anti-cancer agents forcombined use may be radiotherapeutic, chemotherapeutic, anti-angiogenicor apoptosis-inducing agents, cytokines or antibodies or anantibody-therapeutic agent constructs that bind to a tumor cell, anintracellular antigen released from a necrotic tumor cell or to acomponent of tumor vasculature (i.e., anti-cancer immunotoxins orcoaguligands). The term “chemotherapeutic agent”, as used herein,includes genes, vectors, antisense constructs and ribozymes.

[0138] Certain preferred second, anti-cancer agents for combined use arethose that complement or enhance the therapeutic effect of theanti-aminophospholipid or anti-anionic phospholipid antibody orsubstantially cell impermeant PE-binding peptide derivative and/or thoseselected for a particular tumor type or patient. “Therapeutic agentsthat complement or enhance the therapeutic effect” includeradiotherapeutic agents, vascular permeability enhancing agents,anti-angiogenic agents, apoptosis-inducing agents, certain cytokines,anti-tumor cell immunotoxins, as well as selected chemotherapeuticagents. Currently preferred “selected chemotherapeutic agents” arechemotherapeutic agents with anti-angiogenic effects, as in Table E;chemotherapeutic agents that induce apoptosis, as in Table F; calciumflux inducing agents, inflammatory cytokines, H₂O₂, thrombin, andanti-tubulin drugs from the combretastatin family. Doxorubicin,etoposide and actinomycin-D are further preferred, with docetaxel beingmost preferred.

[0139] The invention further provides a liposome, lipid carrier,complex, mixture, supramolecular structure multimolecular aggregate orlipid-based drug delivery system comprising at least a first purifiedanti-aminophospholipid or anti-anionic phospholipid antibody, orantigen-binding fragment or immunoconjugate thereof, preferably one thatbinds to essentially the same epitope as the monoclonal antibody 9D2 or3G4 (ATCC PTA 4545), or a substantially cell impermeant PE-bindingpeptide derivative, preferably a substantially cell impermeant duramycinderivative, or an anti-viral conjugate thereof. The liposome orliposome-like composition may be in the form of a monolayer, bilayer,multimolecular aggregate, vesicle, helix, disc, tube, fiber, torus,hexagonal phase, gel phase, liquid-crystalline phase, liquid-crystallinemultimolecular aggregate, micelle, reverse micelle, microemulsion,emulsion, microreservoir, oil globule, fat globule, wax globule and/orcolloidal particle.

[0140] Liposomes or liposome-like compositions generally comprise an“outer membrane” or bulk aqueous phase and “central core” or inneraqueous phase. In preferred embodiments, the liposome or liposome-likecomposition is a stealthed liposome, lipid carrier, complex, mixture,supramolecular structure multimolecular aggregate or lipid-based drugdelivery system. “Stealthed” liposomes and liposome-like compositionscomprise a biologically effective amount of at least a first stealthingagent in operative association with the outer membrane. A “stealthingagent” is a component that increases the biological half life of aliposome or liposome-like composition when operatively associated withthe outer membrane of the liposome or liposome-like composition. In“operative association”, the outer membrane of the liposome orliposome-like composition is preferably “coated” with the one or morestealthing agents.

[0141] Effective stealthing agents include a range of biocompatiblehydrophilic polymers, such as polyamines, polylactic acid, polyglycolicacid, polylactic-polyglycolic acid (PLGA), polypeptides and relatedmaterials. A preferred stealthing agent is polyethylene glycol (PEG)component, wherein the resulting stealthed liposomes are termed“PEGylated liposomes”.

[0142] Preferred liposomes of the invention are stealthed or PEGylatedliposomes wherein an antibody to an aminophospholipid or anionicphospholipid, or antigen-binding fragment thereof, preferably one thatcompetes with the monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), isoperatively associated with the outer membrane of the liposome,preferably where the liposome is “coated” with the antibody or fragmentthereof.

[0143] Particularly preferred liposomes are such “antibody-coated”stealthed or PEGylated liposomes wherein at least a first therapeuticagent, such as an anti-viral agent or preferably an anti-cancer agent,is operatively associated with the liposome or dispersed within theliposomal formulation. Preferably, the therapeutic, anti-viral oranti-cancer agent is operatively associated with or maintained withinthe central core of the liposome. Exemplary anti-cancer agents areradionuclide(s) and chemotherapeutic agents, such as anti-tubulin drugs,docetaxel and paclitaxel, with docetaxel being preferred.

[0144] For combinations with biological, diagnostic, anti-angiogenic,anti-cancer agents and stealthed liposomes, antibodies that bind tosubstantially the same epitope as, i.e., compete with, the monoclonalantibody 9D2 or 3G4 (ATCC PTA 4545) are particularly preferred. Wherethe invention is intended for use in treating a viral infection ordisease, the at least a second therapeutic agent will preferably be “atleast a second, anti-viral agent or drug”. The invention thus alsoprovides a range of combined anti-viral compositions and formulations,not limited to the 3G4 and like antibodies.

[0145] These aspects of the invention can be conveniently described as acomposition, pharmaceutical composition, combination, mixture,medicament and/or medicinal cocktail comprising at least a firstanti-viral agent or drug in combination with a biologically ortherapeutically effective amount of at least one purifiedanti-aminophospholipid or anti-anionic phospholipid antibody, orantigen-binding fragment or immunoconjugate thereof, optionally one thatbinds to essentially the same epitope as the monoclonal antibody 9D2 or3G4 (ATCC PTA 4545), or a substantially cell impermeant PE-bindingpeptide derivative, preferably a substantially cell impermeant duramycinderivative, or an anti-viral conjugate thereof.

[0146] In the foregoing description, the anti-viral agent or drug isrecited as the “at least a first anti-viral agent or drug” and theantibody, fragment, immunoconjugate, substantially cell impermeantPE-binding peptide, duramycin derivative or an anti-viral conjugatethereof is recited as the second component of the combination. This is amatter of grammatical convenience.

[0147] The one or more anti-viral agents or drugs for use in the presentcombined compositions may be selected from any anti-viral agent or drugavailable at the time of practicing the invention, including the rangeof anti-viral agents and drugs described herein for attachment toantibodies and peptides of the invention. By way of example,anti-retroviral drugs such as NTRIs, non-nucleoside RT inhibitors andprotease inhibitors, anti-viral agents as set forth in Table G, andpreferably, AZT or cidofovir.

[0148] Further embodiments of the invention concern kits comprising, inat least a first composition or container, at least a first purifiedanti-aminophospholipid or anti-anionic phospholipid antibody, orantigen-binding fragment or immunoconjugate thereof, optionally one thatbinds to essentially the same epitope as the monoclonal antibody 9D2 or3G4 (ATCC PTA 4545), or a substantially cell impermeant PE-bindingpeptide derivative, preferably a substantially cell impermeant duramycinderivative, or an anti-viral conjugate thereof, in combination with abiologically or therapeutically effective amount of at least a secondbiological agent, component or system.

[0149] The “second biological agents, components or systems” are notlimited to therapeutic or diagnostic agents. For example, secondbiological agents, components or systems may comprise components formodification of the antibody and/or for attaching other agents to theantibody. Certain preferred second biological agents, components orsystems are prodrugs or components for making and using prodrugs,including components for making the prodrug itself and components foradapting the antibodies of the invention to function in such prodrug orADEPT embodiments.

[0150] The at least a “second diagnostic agent, component or system” maybe a diagnostic agent, component or system directly or indirectlydetectable by an in vitro diagnostic test. “Directly detectable in vitroreporter agents” include radiolabels, reporter agents detectable byimmunofluorescence and luciferase. “Indirectly detectable in vitroreporter agents” function in conjunction with further exogenousagent(s), such as detectable enzymes that yield a colored product oncontact with a chromogenic substrate. These include “secondaryantibodies”, which are attached to a direct or indirect detectableagent, such a radiolabel or enzyme, and “secondary and tertiary antibodydetection systems” in which the tertiary antibody is attached to thedetectable agent.

[0151] Preferred diagnostic kits of the invention are those comprising adiagnostic agent, component or system detectable by in vivo diagnosis orimaging. An advantage of the imaging embodiments of the invention isthat the same antibody can be used for imaging and treatment. Theinvention therefore provides kits and medicaments that comprise:

[0152] (a) a first pharmaceutical composition comprising adiagnostically effective amount of an anti-aminophospholipid oranti-anionic phospholipid antibody, or antigen-binding fragment thereof,preferably one that binds to essentially the same epitope as themonoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), operatively attached toa detectable label or diagnostic agent; and

[0153] (b) a second pharmaceutical composition comprising atherapeutically effective amount of anti-aminophospholipid oranti-anionic phospholipid antibody, or antigen-binding fragment orimmunoconjugate thereof, preferably one that binds to essentially thesame epitope as the monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545).

[0154] For use in therapeutic embodiments, the kits will comprise “atleast a second therapeutic agent”. Preferably, such kits comprise acombined biologically or therapeutically effective amount of at leastthe two specified agents, such as combined amounts effective to inhibitproliferation or viral replication, or to treat a disease such as anangiogenic disease, cancer or a viral infection.

[0155] In terms of cancer treatment, the kits of the invention includeantibodies for use in combination with prodrugs and ADEPT. In suchcompositions, the antibody or fragment thereof is “modified to provide aconverting or enzymatic capacity”. This can be achieved by making acatalytic antibody. Preferably, the antibody is operatively associatedwith, preferably covalently linked or conjugated to, at least a firstconverting agent or enzyme capable of converting at least one prodrug tothe active form of the drug.

[0156] The enzymatic or enzyme-conjugated antibody or fragment willcombined with an initially separate formulation of the “prodrug”. Theprodrug will be an inactive or weakly active form of a drug that is thatis converted to the active form of the drug on contact with theenzymatic capacity, converting function or enzyme associated with theanti-aminophospholipid or anti-anionic phospholipid antibody of theinvention, preferably one that competes with the monoclonal antibody 9D2or 3G4 (ATCC PTA 4545).

[0157] Accordingly, kits are provided that comprise, preferably inseparate compositions and/or containers:

[0158] (a) a biologically effective amount of at least a firstanti-aminophospholipid or anti-anionic phospholipid antibody, orantigen-binding fragment thereof, preferably one that competes with themonoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), which has an enzymaticfunction, preferably where the antibody or fragment is operativelyassociated with, covalently linked or conjugated to, at least a firstenzyme; and

[0159] (b) a biologically effective amount of at least a firstsubstantially inactive prodrug that is converted to a substantiallyactive drug by the enzymatic function of, or by the enzyme associatedwith, linked to or conjugated to the anti-aminophospholipid oranti-anionic phospholipid antibody or fragment thereof.

[0160] Suitable enzymes that cleave a substantially inactive prodrug torelease a substantially active drug include arylsulfatase, serratiaprotease, thermolysin, subtilisin, a carboxypeptidase, a cathepsin,D-alanylcarboxypeptidase, β-galactosidase, neuraminidase, β-lactamase,penicillin amidase and cytosine deaminase.

[0161] Other than prodrugs, the at least a second, anti-cancer agent maybe any of the second, anti-cancer agents described above in relation tothe combined anti-cancer compositions of the invention. For treatingviral infections, the at least a second, anti-viral agent may also beany of the second, anti-viral agents described above in relation to thecombined anti-viral compositions of the invention. However, the “kits”may comprise the at least two recited the agents “in combination, butseparately”, thus providing even more flexibility in the selection ofagents.

[0162] The kits of the invention may therefore comprise combinedbiologically or therapeutically effective amounts of at least the twospecified agents within a single container or container means, or withindistinct containers or container means. The kits may also compriseinstructions for using the biological and therapeutic agents includedtherein. Imaging components may also be included in combination, butseparately with the therapeutic kits.

[0163] Anti-Angiogenic and Tumor Treatment: The present inventionprovides a number of methods and uses of the anti-aminophospholipid oranti-anionic phospholipid antibodies, including the 9D2- and 3G4-likeantibodies, and the substantially cell impermeant PE-binding peptide andduramycin derivatives. Concerning all methods, the terms “a” and “an”are used to mean “at least one”, “at least a first”, “one or more” or “aplurality” of steps in the recited methods, except where specificallystated. This is particularly relevant to the administration steps in thetreatment methods. Thus, not only may different doses be employed withthe present invention, but different numbers of doses, e.g., injectionsor inhalations, may be used, up to and including multiple injections orinhalations.

[0164] Various useful in vitro methods and uses are provided that haveimportant biological implications. First provided are methods of, anduses in, binding aminophospholipids or anionic phospholipids, preferablyPS or PE, which generally comprise effectively contacting a compositioncomprising an aminophospholipid or anionic phospholipid, preferably PSor PE, with at least a first anti-aminophospholipid or anti-anionicphospholipid antibody, or antigen-binding fragment thereof, preferablyan antibody that binds to substantially the same epitope as themonoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), or with a substantiallycell impermeant duramycin derivative. The “contacting” is underconditions effective to allow the formation of bound complexes, and anycomplexes so formed are detected. The detection methods and uses may beused in connection with biological samples, e.g., in diagnostics forapoptosis, tumors and virally infected cells, and diagnostic kits basedthereon are also provided.

[0165] Proliferation inhibition methods and uses are provided, whichpreferably use the antibodies, antigen binding fragments andimmunoconjugates of the invention. Methods to inhibit endothelial cellproliferation and/or migration generally comprise contacting apopulation of cells or tissues that includes a population of endothelialcells with a composition comprising a biologically effective amount ofat least a first anti-aminophospholipid or anti-anionic phospholipidantibody, optionally one that binds to substantially the same epitope asthe monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), or anantigen-binding fragment thereof, under conditions effective to inhibitendothelial cell proliferation and/or migration.

[0166] The foregoing methods and uses can be performed in vitro and invivo, in the latter case, wherein the tissues or cells are locatedwithin an animal and the anti-aminophospholipid or anti-anionicphospholipid antibody is administered to the animal. In both cases, themethods and uses become methods and uses for inhibiting angiogenesis,comprising contacting a population of potentially angiogenic bloodvessels, or a tissue comprising a population of potentially angiogenicblood vessels, with an anti-angiogenic composition comprising abiologically effective amount of at least a first anti-aminophospholipidor anti-anionic phospholipid antibody, optionally one that binds tosubstantially the same epitope as the monoclonal antibody 9D2 or 3G4(ATCC PTA 4545), or an antigen-binding fragment thereof, underconditions effective to inhibit angiogenesis.

[0167] Where populations of potentially angiogenic blood vessels aremaintained ex vivo, the present invention has utility in drug discoveryprograms. In vitro screening assays, with reliable positive and negativecontrols, are useful as a first step in the development of drugs toinhibit or promote angiogenesis, as well as in the delineation offurther information on the angiogenic process. Where the population ofpotentially angiogenic blood vessels is located within an animal orpatient, the anti-angiogenic composition is administered to the animalas a form of therapy.

[0168] Anti-angiogenic and anti-vascular therapies are provided in termsof animals and patients that have, or are at risk for developing, anydisease or disorder characterized by undesired, inappropriate, aberrant,excessive and/or pathological vascularization. It is well known to thoseof ordinary skill in the art that as aberrant angiogenesis occurs in awide range of diseases and disorders, a given anti-angiogenic therapy,once shown to be effective in any acceptable model system, can be usedto treat the entire range of diseases and disorders connected withangiogenesis.

[0169] The methods and uses of the present invention are particularlyintended for use in animals and patients that have, or are at risk fordeveloping, any form of vascularized tumor; macular degeneration,including age-related macular degeneration; arthritis, includingrheumatoid arthritis; atherosclerosis and atherosclerotic plaques;diabetic retinopathy and other retinopathies; thyroid hyperplasias,including Grave's disease; hemangioma; neovascular glaucoma; andpsoriasis.

[0170] As disclosed in U.S. Pat. Nos. 5,712,291 and 6,524,583,specifically incorporated herein by reference, each of the foregoingtreatment groups are by no means exhaustive of the types of conditionsthat are to be treated by the present invention. U.S. Pat. Nos.5,712,291 and 6,524,583 are incorporated herein by reference for certainspecific purposes, including the purpose of identifying a number ofother conditions that may be effectively treated by an anti-angiogenictherapeutic; the purpose of showing that the treatment of all angiogenicdiseases represents a unified concept, once a defined category ofangiogenesis-inhibiting compounds have been disclosed and claimed (inthe present case, anti-aminophospholipid or anti-anionic phospholipidantibodies, optionally those that bind to substantially the same epitopeas the monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545)); and the purposeof showing that the treatment of all angiogenic diseases is enabled bydata from only a single model system.

[0171] In addition to the treatment of angiogenic and vascular diseases,important and unified aspects of the present invention are compositionsand methods for treating cancer. Such methods comprise administering toan animal or patient that has, or is at risk for developing, cancer, abiologically or therapeutically effective amount of at least a firstcomposition comprising at least a first purified anti-aminophospholipidor anti-anionic phospholipid antibody, or antigen-binding fragment orimmunoconjugate thereof, preferably one that binds to essentially thesame epitope as, or competes with, the monoclonal antibody 9D2 or 3G4(ATCC PTA 4545), or a substantially cell impermeant PE-binding peptidederivative, preferably a substantially cell impermeant duramycinderivative.

[0172] The cancer treatment methods of the invention, even those usingthe antibodies, do not rely solely on exerting anti-vascular and/oranti-angiogenic effects. The cancer treatment methods and uses of theinvention are suitable for treating all forms of cancer, includinganimals and patients that have, or are at risk for developing, avascularized solid tumor, a metastatic tumor or metastases from aprimary tumor. The methods of the invention preferably exert ananti-cancer effect without causing significant thrombotic complications.

[0173] Both unconjugated or naked antibodies, and fragments thereof, andimmunoconjugates may be used in the cancer treatment aspects of theinvention. As to the use of immunoconjugates, the invention providesmethods for delivering selected therapeutic or diagnostic agents totumors. Such embodiments comprise administering to an animal or patienthaving a tumor a biologically effective amount of a compositioncomprising at least a first immunoconjugate in which a diagnostic ortherapeutic agent is operatively attached to an anti-aminophospholipidor anti-anionic phospholipid antibody, or antigen-binding fragmentthereof, preferably one that binds to substantially the same epitope as,or competes with, the monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545).

[0174] The invention therefore provides tumor diagnostic, prognostic,imaging and related methods using an anti-aminophospholipid oranti-anionic phospholipid antibody, or antigen-binding fragment thereof,preferably one that binds to substantially the same epitope as, orcompetes with, the monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), todetect pre-apoptotic and apoptotic cells. Such methods can be used as asurrogate marker to monitor the progress of other treatment,particularly chemotherapy, or to form an image of a tumor prior totreatment.

[0175] The use of the invention as a surrogate marker to monitor theprogress of cancer treatment, particularly chemotherapy, comprises:

[0176] (a) subjecting an animal or patient with a tumor to at least afirst treatment designed to exert an anti-tumor effect; and

[0177] (b) subsequently administering to the same animal or patient adiagnostically effective amount of at least a firstanti-aminophospholipid or anti-anionic phospholipid antibody, orantigen-binding fragment thereof, preferably one that binds tosubstantially the same epitope as, or competes with, the monoclonalantibody 9D2 or 3G4 (ATCC PTA 4545), operatively attached to adetectable label or diagnostic agent, thereby forming a detectable imageof the tumor, preferably an image of pre-apoptotic or apoptotic tumorcells or tumor vascular endothelial cells within the tumor; andpreferably

[0178] (c) analyzing the detectable image of the tumor, preferably theimage of the pre-apoptotic or apoptotic tumor cells or tumor vascularendothelial cells within the tumor, thereby assessing the progress oreffectiveness of the at least a first treatment designed to exert ananti-tumor effect.

[0179] The combined imaging and cancer treatment methods comprise:

[0180] (a) forming an image of a tumor by administering to an animal orpatient having a tumor a diagnostically minimal or effective amount ofat least a first anti-aminophospholipid or anti-anionic phospholipidantibody, or antigen-binding fragment thereof, preferably one that bindsto substantially the same epitope as, or competes with, the monoclonalantibody 9D2 or 3G4 (ATCC PTA 4545), operatively attached to adetectable label or diagnostic agent, thereby forming a detectable imageof the tumor; and

[0181] (b) subsequently administering to the same animal or patient atherapeutically optimized or effective amount of at least a firstanti-aminophospholipid or anti-anionic phospholipid antibody, orantigen-binding fragment or immunoconjugate thereof, preferably one thatbinds to essentially the same epitope as, or competes with, themonoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), thereby causing ananti-tumor effect.

[0182] For use in the cancer treatment methods of the invention, thecurrently preferred antibodies are those that bind to substantially thesame epitope as, or compete with, the monoclonal antibody 9D2 and 3G4(ATCC PTA 4545). In terms of immunoconjugates, anti-aminophospholipid oranti-anionic phospholipid antibodies, preferably those that compete withthe monoclonal antibody 9D2 and 3G4 (ATCC PTA 4545), linked to ananti-cancer agent from Table E or Table F, a combretastatin, gelonin,TNFα, IL-12 and LEC are currently preferred. The currently preferredsubstantially cell impermeant PE-binding peptide derivatives use incancer treatment are duramycin derivatives, most preferably duramycinlinked to biotin or duramycin linked to HIgG.

[0183] Within the antibody-based cancer treatment methods of theinvention, the invention further provides prodrug treatment methods,which generally comprise:

[0184] (a) administering to an animal or patient with a tumor a firstpharmaceutical composition comprising a first anti-aminophospholipid oranti-anionic phospholipid antibody, or antigen-binding fragment thereof,preferably one that competes with the monoclonal antibody 9D2 or 3G4(ATCC PTA 4545), which antibody or fragment thereof has an enzymaticfunction, preferably where the antibody or fragment is operativelyassociated with, covalently linked or conjugated to, at least a firstenzyme; wherein the antibody or fragment localizes to the tumor afteradministration and

[0185] (b) subsequently administering to the animal or patient, after aneffective time period, at least a second pharmaceutical compositioncomprising a biologically effective amount of at least one substantiallyinactive prodrug; wherein the prodrug is converted to a substantiallyactive drug by the enzymatic function of, or by the enzyme associatedwith, linked to or conjugated to the anti-aminophospholipid oranti-anionic phospholipid antibody, or fragment thereof, localizedwithin the tumor.

[0186] The present invention further provides a range of combinationcancer treatment methods, comprising administering to an animal orpatient with cancer a therapeutically effective combined amount of atleast a first purified anti-aminophospholipid or anti-anionicphospholipid antibody, or antigen-binding fragment or immunoconjugatethereof, optionally one that binds to essentially the same epitope asthe monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), or a substantiallycell impermeant PE-binding peptide derivative, preferably asubstantially cell impermeant duramycin derivative, and at least asecond, distinct therapeutic or anti-cancer agent.

[0187] Generally speaking, the at least a second anti-cancer agent maybe administered to the animal or patient before, during or afteradministration of the anti-aminophospholipid or anti-anionicphospholipid antibody, 9D2- or 3G4-based therapeutic or substantiallycell impermeant duramycin derivative. The at least a second anti-canceragent may be administered to the animal or patient “substantiallysimultaneously” with the anti-aminophospholipid or anti-anionicphospholipid antibody, 9D2- or 3G4-based therapeutic or substantiallycell impermeant duramycin derivative; such as from a singlepharmaceutical composition or from two pharmaceutical compositionsadministered closely together.

[0188] Alternatively, the at least a second anti-cancer agent may beadministered to the animal or patient at a time sequential to theadministration of the anti-aminophospholipid or anti-anionicphospholipid antibody, 9D2- or 3G4-based therapeutic or substantiallycell impermeant duramycin derivative. “At a time sequential”, as usedherein, means “staggered”, such that the at least a second anti-canceragent is administered to the animal or patient at a time distinct to theadministration of the anti-aminophospholipid or anti-anionicphospholipid antibody, 3G4-based therapeutic or substantially cellimpermeant duramycin derivative.

[0189] In sequential administration, the two agents are administered attimes effectively spaced apart to allow the two agents to exert theirrespective therapeutic effects, i.e., they are administered at“biologically effective time intervals”. The at least a secondanti-cancer agent may be administered to the animal or patient at abiologically effective time prior to the anti-aminophospholipid oranti-anionic phospholipid antibody, 9D2- or 3G4-based therapeutic orsubstantially cell impermeant duramycin derivative, or at a biologicallyeffective time subsequent to that therapeutic.

[0190] Any therapeutic or anti-cancer agent may be used as the second,therapeutic or anticancer agent in the combined cancer treatment methodsof the invention, including any of the therapeutic or anti-cancer agentsdescribed above in relation to the anti-cancer compositions and kits ofthe invention. Preferred agents are those that complement or enhance thetherapeutic effects of the antibodies, fragments, immunotoxins orpeptide derivatives, such as vascular permeability enhancing agents,anti-angiogenic agents, apoptosis-inducing agents, calcium flux inducingagents, inflammatory cytokines, antibodies and immunotoxins to tumorcells and necrotic tumor cells, chemotherapeutic agents from Table E orTable F, a combretastatin, doxorubicin, etoposide and actinomycin-D.

[0191] Docetaxel is a particularly preferred agent for use incombination therapy. Docetaxel may be administered separately to theanti-aminophospholipid or anti-anionic phospholipid antibody,substantially cell impermeant PE-binding peptide or duramycinderivative, either before or afterwards. As to simultaneousadministration, docetaxel may be given in separate or the sameformulations, optionally within a liposome or stealthed liposome, andpreferably within the core of a stealthed liposome coated with anantibody that binds to an aminophospholipid or anionic phospholipid,preferably an antibody that binds to essentially the same epitope as, orcompetes with, the monoclonal antibody 9D2 or 3G4 (ATCC PTA 4545).

[0192] Treating Viral Infections: Particularly important and surprisingdevelopments of the invention concern antibodies, immunoconjugates,peptides, peptide conjugates, compositions, combinations, kits, methods,uses and medicaments for inhibiting viruses and for treating orpreventing viral infections. In a first instance, the anti-viral methodsof the invention concern contacting a composition comprising, orpopulation of cells or tissue(s) that contains or is suspected tocontain, a virally infected cell with at least a first compositioncomprising a biologically effective amount of at least a first purifiedantibody that binds to an aminophospholipid or anionic phospholipid,preferably to PS or PE, optionally one that binds to essentially thesame epitope as, or competes with, the monoclonal antibody 9D2 or 3G4(ATCC PTA 4545), or antigen-binding fragment, immunoconjugate oranti-viral conjugate thereof, or a substantially cell impermeantPE-binding peptide derivative, preferably a substantially cellimpermeant duramycin derivative, or an anti-viral conjugate thereof. Thevirally infected cell is preferably a eukaryotic cell, such as an animalcell, and preferably a mammalian or human cell.

[0193] The anti-viral methods and uses can be performed in vitro and invivo. In the in vitro embodiments, the methods have important utilities.For example, in drug discovery programs for the development ofanti-viral drugs or combinations thereof, as well as in the delineationof further information on viral infection, replication and spread. Thein vitro anti-viral methods may also be used in purging viruses frombiological samples, such as cell populations and tissue cultures forlaboratory use, from samples, tissues, seeds, plant parts and plants foragricultural use, and from blood and tissue samples for therapeutic use.In the in vivo methods, where the cells, populations or tissues arelocated within an animal, the anti-aminophospholipid or anti-anionicphospholipid antibody, fragment, immunoconjugate, substantially cellimpermeant PE-binding peptide, duramycin derivative or anti-viralconjugate thereof, is administered to the animal as anti-viral therapy.

[0194] In both cases, the compositions, methods and uses inhibit one ormore steps or stages necessary for a productive or ongoing viralinfection, including inhibiting viral entry. Preferably, thecompositions, methods and uses inhibit viral replication and/or spread,such as inhibiting one or more steps of viral transcription,translation, assembly, packaging and/or egress within or from aninfected host cell, such as a mammalian or human cell. The inventiontherefore preferably limits or substantially confines viral infectionsto initially infected cells and cell populations, thus substantiallyinhibiting or preventing the subsequent or ongoing infection ofadditional host cells or tissues.

[0195] The anti-viral treatment methods of the invention preferablyconcern administering to an animal or patient having, suspected ofhaving or at risk for developing a viral infection or associated diseaseat least a first composition comprising a biologically effective amountof at least a first purified antibody that binds to an aminophospholipidor anionic phospholipid, preferably to PS or PE, optionally one thatbinds to essentially the same epitope as, or competes with, themonoclonal antibody 9D2 or 3G4 (ATCC PTA 4545), or antigen-bindingfragment, immunoconjugate or anti-viral conjugate thereof, or asubstantially cell impermeant PE-binding peptide derivative, preferablya substantially cell impermeant duramycin derivative, or an anti-viralconjugate thereof.

[0196] Currently preferred therapeutic agents for use in anti-viraltreatment are antibodies that bind to an aminophospholipid, preferablyto PS or PE, and immunoconjugates of such antibodies operativelyattached to at least a second, distinct anti-viral agent; duramycinpeptides and derivatives linked to biotin or linked to HIgG, andconjugates of PE-binding peptides, preferably duramycins, operativelylinked to at least a second, distinct anti-viral agent. Suitableanti-viral agents for attachment to the antibodies and peptides includethose set forth in Table G, such as AZT or cidofovir.

[0197] As the invention inhibits one or more steps or stages necessaryfor productive or ongoing infection common to all viruses, theanti-viral methods and uses of the invention are suitable for treatingall viruses, both enveloped and non-enveloped viruses, including thosethat infect plants, animals, vertebrates, mammals and human patients.The invention is suitable for treating all viruses that infectvertebrates, as listed herein in Table H, particularly humans, andparticularly viruses that are pathogenic in animals and humans. Theviral infections and associated and resultant diseases that can betreated by the invention include those viruses and diseases set forth inTable J, as exemplified by treating CMV, RSV, arenavirus and HIVinfections, and the diseases hepatitis, influenza, pneumonia, Lassafever and AIDS.

[0198] The anti-viral treatment methods of the invention may also beused in combination with other therapeutics and diagnostics. Thecombined treatment methods comprise administering to an animal orpatient with a viral infection a therapeutically effective combinedamount of at least a first composition comprising at least a firstpurified antibody that binds to an aminophospholipid or anionicphospholipid, preferably to PS or PE, optionally one that binds toessentially the same epitope as, or competes with, the monoclonalantibody 9D2 or 3G4 (ATCC PTA 4545), or antigen-binding fragment,immunoconjugate or anti-viral conjugate thereof, or a substantially cellimpermeant PE-binding peptide derivative, preferably a substantiallycell impermeant duramycin derivative, or an anti-viral conjugatethereof, and at least a second, distinct therapeutic or anti-viralagent.

[0199] The at least a “second, distinct” anti-viral agent is inreference to the anti-aminophospholipid or anti-anionic phospholipidantibody, fragment or immunoconjugate, or substantially cell imperneantPE-binding peptide, duramycin derivative or anti-viral conjugatethereof, being the “first” anti-viral agent. The at least a secondanti-viral agent may be administered to the animal or patient duringadministration of, or substantially simultaneously with, the firstanti-viral agent of the invention; or before or after, i.e., sequentialto the administration of the first anti-viral agent of the invention.

[0200] Any therapeutic or anti-viral agent may be used as the secondtherapeutic or anti-viral agent in the combined anti-viral treatmentmethods of the invention, including any of the anti-viral agentsdescribed above in relation to the anti-viral conjugates, compositionsand kits of the invention.

[0201] The foregoing cancer and anti-viral treatment methods and useswill often involve the administration of the pharmaceutically effectivecomposition to the animal or patient systemically, such as bytransdermal, intramuscular, intravenous injection and the like. Fortreating viral infections, particularly respiratory viral infections,delivery to the lung is another preferred embodiment, as may be achievedusing an aerosol. However, any route of administration that allows thetherapeutic agent to localize to the site of the tumor or viralinfection will be acceptable. Therefore, other suitable routes ofdelivery include oral, rectal, nasal, topical, and vaginal. For uses andmethods for the treatment of arthritis, e.g., intrasynovialadministration may be employed, as described for other immunologicalagents in U.S. Pat. No. 5,753,230, specifically incorporated herein byreference. For conditions associated with the eye, ophthalmicformulations and administration are contemplated.

[0202] “Administration”, as used herein, means provision or delivery ofanti-aminophospholipid or anti-anionic phospholipid antibody or 9D2- or3G4-based therapeutics, or substantially cell impermeant PE-bindingpeptide derivatives, preferably duramycin derivatives in an amount(s)and for a period of time(s) effective to exert a therapeutic effect. Thepassive administration of proteinaceous therapeutics is generallypreferred, in part, for its simplicity and reproducibility.

[0203] However, the term “administration” is herein used to refer to anyand all means by which the therapeutics are delivered. “Administration”therefore includes the provision of cells that produce theanti-aminophospholipid or anti-anionic phospholipid antibody, 3G4-basedor duramycin derivative therapeutics in an effective manner. In suchembodiments, it may be desirable to formulate or package the cells in aselectively permeable membrane, structure or implantable device,generally one that can be removed to cease therapy. Exogenousadministration will still generally be preferred, as this represents anon-invasive method that allows the dose to be closely monitored andcontrolled.

[0204] The therapeutic methods and uses of the invention also extend tothe provision of nucleic acids that encode anti-aminophospholipid oranti-anionic phospholipid antibody, 3G4-based or duramycin derivativetherapeutics in a manner effective to result in their expression invivo. Any gene therapy technique may be employed, such as naked DNAdelivery, recombinant genes and vectors, cell-based delivery, includingex vivo manipulation of patients' cells, and the like. Liposomes andstealthed liposomes will be preferred for use in some embodiments.

[0205] The pharmaceutical compositions and treatment methods of theinvention employ “therapeutically effective amounts” of ananti-aminophospholipid or anti-anionic phospholipid antibody, optionallyone that binds to substantially the same epitope as the monoclonalantibody 9D2 or 3G4 (ATCC PTA 4545), or an antigen-binding fragment orimmunoconjugate of such an antibody, or a substantially cell impermeantPE-binding peptide derivative, preferably a substantially cellimpermeant duramycin derivative, or an anti-viral conjugate thereof. The“therapeutic effects” and consequent “therapeutically effective amounts”are measured by different parameters in cancer treatment vs. anti-viraltreatment.

[0206] In cancer treatment, the amounts of the agents are effective tospecifically kill at least a portion of tumor cells, tumor orintratumoral vascular endothelial cells; to specifically induceapoptosis in at least a portion of tumor cells, tumor or intratumoralvascular endothelial cells; to specifically promote coagulation in atleast a portion of tumor or intratumoral blood vessels; to specificallyocclude or destroy at least a portion of blood transporting vessels ofthe tumor; to specifically induce necrosis in at least a portion of atumor; and/or to induce tumor regression or remission uponadministration to an animal or patient.

[0207] In treating viral infections and related diseases, the amounts ofthe agents are effective to inhibit one or more requirements for ongoingviral infection, such as viral entry, and preferably, viral replication,egress and spread from the infected host cells. The amounts may alsokill or remove at least a portion of the virally infected cells in amanner that counteracts viral replication, spread and ongoing infection.Overall, the amounts of the agents are effective to reduce,significantly reduce or eradicate the viral infection uponadministration to an animal or patient.

[0208] The terms “preferentially” and “specifically”, as used herein,mean that the anti-aminophospholipid or anti-anionic phospholipidantibody, 3G4-based therapeutics, or substantially cell impermeantPE-binding peptide derivatives, preferably duramycin derivatives,achieve anti-cancer or anti-viral effects that are substantiallyconfined to the disease site, and do not substantially causecoagulation, destruction and/or tissue necrosis in normal, healthytissues of the animal or subject. The structure and function of healthycells and tissues is therefore maintained substantially unimpaired bythe practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0209] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein. The U.S. file of this patentcontains at least one drawing executed in color. Copies of this patentwith color drawing(s) will be provided by the Patent and TrademarkOffice upon request and payment of the necessary fee.

[0210]FIG. 1. Localization of anti-PS antibody (3SB) to vascularendothelial cells in L540 human Hodgkin's lymphoma, 3LL murine lungcarcinoma and B16 murine melanoma tumors in mice. Tumor-bearing SCIDmice were injected intravenously with 20 μg of anti-PS (3SB) or anti-CL(D11) mouse IgM. The blood circulation was perfused with saline 1 hlater. Mice were sacrificed 1 h later and tumor and organs wereharvested and snap-frozen. Mouse IgM was detected on frozen sectionsusing goat anti-mouse IgM-peroxidase conjugate. Anti-PS antibodyspecifically localized to blood vessels (indicated by arrows) in alltumors. No localization was observed in mice injected with control,anti-CL IgM.

[0211]FIG. 2A and FIG. 2B. Binding of 9D2 antibody and annexin V tophospholipids adsorbed to plastic. Phospholipids were adsorbed toplastic of microtiter plates. After blocking with 10% serum, 9D2antibody (FIG. 2A) or annexin V (FIG. 2B) were added at concentrationsranging from 6.66 nM to 0.005 nM in the presence of 10% serum. Theplates were washed and the bound 9D2 antibody and annexin V weredetected using goat anti-rat IgM-HRP and rabbit anti-annexin V IgGfollowed by anti-rabbit-HRP, respectively.

[0212]FIG. 3. Inhibition of binding of 9D2 antibody and annexin V toanionic phospholipids on H₂O₂-treated endothelial cells with competingphospholipid liposomes. 9D2 antibody and annexin V (6.66 nM) werepre-incubated with various phospholipid liposomes (200 μg/ml) DPBSbuffer containing 10% serum. The bound 9D2 antibody and annexin V weredetected using goat anti-rat IgM-HRP and rabbit anti-annexin V IgGfollowed by anti-rabbit-HRP respectively. Binding in the presence orabsence of competing liposomes was determined. Standard deviations oftriplicate measurements were less than 10% of the mean values.

[0213]FIG. 4. Localization of biotinylated 9D2 antibody and annexin V tovascular endothelial cells and tumor cells in orthotopic MDA-MB-231human breast tumors in mice. Nu/nu mice bearing MDA-MB-231 tumors intheir mammary fat pads were injected intravenously with 50 μg ofbiotinylated 9D2 antibody or 100 μg of biotinylated annexin V. One hlater, their blood circulation was perfused with saline. Tumor andorgans were removed and snap-frozen. Localized 9D2 and annexin V weredetected on the frozen sections using streptavidin-HRP conjugate. Tumorsections derived from mice injected with saline or control rat IgMserved as negative controls.

[0214]FIG. 5. Combined effects of hypoxia and inflammatory cytokines onPS exposure. bEnd.3 cells were treated for 24 h with IL-1α and TNFαunder normoxic (white bars) and hypoxia (gray bars) conditions. The cellmonolayers remained intact and viable under these conditions. PSexternalization was determined by measuring binding of ¹²⁵I-annexin V.The level of PS exposure was expressed as a percentage of that in cellstreated with combination of actinomycin D and TNFα.

[0215]FIG. 6A and FIG. 6B. Anti-tumor effects of anti-PS antibody (3SB)in animals with syngeneic and xenogeneic tumors. 1×10⁷ cells of murinecolorectal carcinoma Colo 26 (FIG. 6A) or human Hodgkin's lymphoma L540(FIG. 6B) were injected subcutaneously into the right flank of BALB/cmice (FIG. 6A) or male CB17 SCID mice (FIG. 6B), respectively. Tumorswere allowed to grow to a size of about 0.6-0.9 cm³ and then the mice (4animals per group) were injected i.p. with 20 μg of naked anti-PSantibody (open squares) or saline (open circles). Treatment was repeated3 times with a 48 hour interval. Animals were monitored daily for tumormeasurements and body weight. Mice were sacrificed when tumors hadreached 2 cm³, or earlier if tumors showed signs of necrosis orulceration. Control mouse IgM gave similar results to saline.

[0216]FIG. 7. Anti-tumor effects of 9D2 antibody in mice bearing L540human Hodgkin's lymphoma. Groups of tumor-bearing mice were injectedwith 100 μg of 9D2 antibody (closed circles) intraperitoneally 3 timesper week, as opposed to control (open squares). The tumor size was takenby calipers twice a week. The tumor volume is plotted against the numberof days after tumor cell injections. The numbers in parentheses indicatenumber of mice with regressed tumors/total number of mice per group.

[0217]FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E, FIG. 8F and FIG. 8G.Anti-tumor effects of anti-PS antibody, 3G4, in animals with syngeneicand xenogeneic tumors. Cells of murine Meth A tumors (FIG. 8A), humanMDA-MB-231 breast cancer (FIG. 8B and FIG. 8E), human Hodgkin's lymphomaL540 (FIG. 8C and FIG. 8D) and MDA-MB-231 cancer (FIG. 8F and FIG. 8G)were injected into mice. Tumors were allowed to grow to the sizes shownbefore treatment. The human Hodgkin's lymphoma cells were allowed toform large tumors. Each group of mice was injected intraperitoneally 3times per week with 100 μg of 3G4 antibody as opposed to control (3G4 isstated on FIG. 8A, FIG. 8B, FIG. 8C; and shown by open circles on FIG.8D, FIG. 8E, FIG. 8F). Animals were monitored twice a week for tumormeasurements. The tumor volume is plotted against the number of daysafter tumor inoculation (FIG. 8A) or against the days of treatment (FIG.8B and FIG. 8C) for 20-30 days (FIG. 8A, FIG. 8B and FIG. 8C; numbers inparentheses indicate number of mice with regressed tumors/total numberof mice per group) or 60 days (FIG. 8D, FIG. 8E and FIG. 8F). The 3G4antibody and chimeric 3G4 antibody (ch3G4) were used to treat MDA-MB-231cancer cells, as opposed to control (FIG. 8G).

[0218]FIG. 9A and FIG. 9B. Inhibition of CMV replication in vitro by 3G4antibody. CMV-infected HHF-R2 cells were treated with 3G4 (top twopanels). The control wells were left untreated (bottom two panels) orwere treated with the isotype matched control IgG₃ antibody GV39G(middle two panels). Cells were observed at different time points: day 3(left column) and day 9 (right column). Infected cells appear greenunder the fluorescent microscope. Antibody treatment at 100 μg/ml (FIG.9A) and 50 μg/ml (FIG. 9B).

[0219]FIG. 10. Concentration dependent inhibition of CMV replication invitro. CMV-infected HHF-R2 cells were treated with differentconcentrations of 3G4 (top panels). The control wells were leftuntreated (bottom panel) or were treated with the isotype matchedcontrol IgG₃ antibody GV39G (middle panels). Cells were observed on day9. Infected cells appear green under the fluorescent microscope.

[0220]FIG. 11A, FIG. 11B and FIG. 11C. Quantification of CMV viral loadin antibody-treated cells and inhibition of replication at a late stageof the viral replication cycle. Monolayers of human fibroblasts wereinfected with CMV at a low m.o.i. of 0.01 pfu/cell and treated with theindicated concentrations of the 3G4 antibody; the control antibody,GV39G; or the control anti-colchicine antibody, C44 (FIG. 11A; Untreat.,untreated control). Monolayers of human fibroblasts were infected withCMV at a high m.o.i. of 3 pfu/cell and treated with 50 μg/ml or 100μg/ml of the 3G4 antibody or the control antibody, GV39G (FIG. 11B).Monolayers of human fibroblasts were infected with CMV at a high m.o.i.,the 3G4 antibody or the control antibody, GV39G were added at theindicated time points after infection (FIG. 11C). In each of FIG. 11A,FIG. 11B and FIG. 11C, the viral load in cells and supernatants wasquantified using a standard plaque assay.

[0221]FIG. 12. Inhibition of RSV replication in vitro by 3G4, 1B9 and3SB antibodies. RSV-infected A-549 cells were treated with 3G4, 1B9 or3SB or left untreated as control. Treatment with 1B9 (green) and 3SB(red) resulted in a log decrease in viral replication (vs. control inblue). The even more pronounced anti-viral effect of 3G4 is shown inpink.

[0222]FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E, FIG. 13F, FIG.13G, FIG. 13H, FIG. 13I, FIG. 13J, FIG. 13K, FIG. 13L, FIG. 13M, FIG.13N, FIG. 13O, FIG. 13P, FIG. 13Q and FIG. 13R. Structures of duramycinderivatives. The chemical structures for exemplary duramycin derivativesfrom Example XV are depicted. In each of the compounds of FIG. 13A toFIG. 13O, the PE-binding peptide, duramycin, has been attached to a cellimpermeant group to prevent the construct from exerting significant,non-specific toxic effects. The schematic structure of the parentduramycin cyclic peptide is shown in FIG. 13P. The linear sequence isrepresented by SEQ ID NO:9, and the structures of the modified aminoacids in the sequence are depicted in FIG. 13Q. FIG. 13R depicts anexemplary duramycin anti-viral construct, in which duramycin is linkedto cidofovir.

[0223]FIG. 14A, FIG. 14B, FIG. 14C and FIG. 14D. Binding specificitiesof duramycin derivatives. The duramycin derivatives were prepared asdescribed in Example XV and their specificities determined using ELISAsand competition ELISAs, as described in Example XVI. FIG. 14A,phospholipid binding profile of duramycin derivatives against a panel ofphospholipids, showing specificity for PE; FIG. 14B, serum has nosignificant effect on PE binding; FIG. 14C and FIG. 14D, results fromcompetition ELISAs confirming specificity of duramycin derivatives forPE.

[0224]FIG. 15. Inhibition of CMV replication in vitro by duramycinderivatives. CMV infected HHF-R2 cells were treated with duramycinderivatives (DLB)₄NA and (DIM)_(n)HIgG. The control wells were leftuntreated. Cells were observed at different time points: day 4 (leftpanels) and day 6 (right panels). Infected cells appear green under thefluorescent microscope. (DLB)₄NA and (DIM)_(n)HIgG inhibit viral spreadfrom singly-infected cells.

[0225]FIG. 16. Selective inhibition of dividing endothelial cells byanti-PS antibodies. The anti-PS antibodies 3SB, 9D2 and 3G4 were testedfor inhibitory effects on endothelial cells in vitro as in ExampleXVIII. Each of the 3SB, 9D2 and 3G4 antibodies exhibit selectiveinhibition of dividing (subconfluent) endothelial cells as opposed toquiescent (confluent) cells. The 9D2 and 3G4 antibodies both have agreater inhibitory effect than 3SB.

[0226]FIG. 17A and FIG. 17B. Anti-angiogenic and vascular targetingeffects of the 3G4 antibody in tumor-bearing mice. Nude mice bearingMDA-MB-231 orthotopic tumors were treated 3 times a week with 100μg/dose 3G4 antibody (treated, right panels) or with the same dose of anisotype-matched, control antibody (control, left panels). At theconclusion of treatment, animals were perfused and tumors weresnap-frozen, cut and stained with an antibody to murine CD31 (rat,anti-mouse CD31), a pan-endothelial marker of murine vasculature (FIG.17A), or embedded in paraffin and strained with H&E (FIG. 17B).Comparing the tumor sections from the control and treated animals showsthat the administration of the 3G4 results in anti-angiogenic (FIG. 17A)and vascular targeting (FIG. 17B) effects.

[0227]FIG. 18A and FIG. 18B. DNA and amino acid sequences of thecomplementarity determining regions (CDRs) of the 3G4 antibody. DNA andamino acid sequences for the heavy (FIG. 18A; SEQ ID NO:1 and SEQ IDNO:2) and light (FIG. 18B; SEQ ID NO:3 and SEQ ID NO:4) chains arepresented, and the restriction sites in the DNA sequences are shown. Theleader sequence is distinguished from the mature protein, which beginsas shown by the first arrow in each of FIG. 18A and FIG. 18B. Exemplarymeans of grafting each variable sequence with a human constant regionare set forth, wherein the first part of the respective human constantregion sequences (SEQ ID NO:7 and SEQ ID NO:8) is shown by the secondarrow in each of FIG. 18A and FIG. 18B.

[0228]FIG. 19A and FIG. 19B. Comparison of the PS binding of the IgGanti-PS antibody, 3G4, with the IgM anti-PS antibody, 3SB. The PSbinding of the IgM antibody, 3SB (♦) and two IgG antibodies, 3G4 (▴) and3B10 (▪), was determined by ELISA using antibody concentrations up to3.375 nM (FIG. 19A). The PS binding of the 3SB (♦), 3G4 (▴) and 3B 10(▪) antibodies at concentrations of up to 0.06 nM is shown separately(FIG. 19B).

[0229]FIG. 20. Inhibition of binding of 3G4 antibody to immobilized PSusing competing phospholipid liposomes. The 3G4 antibody (0.1 μg/ml) waspre-incubated for 30 minutes with various liposomes made from purephospholipids (PS-L, PE-L, PI-L, PC-L, CL-L, PA-L and PG-L) or bufferalone (control). The mixtures were then added to PS-coated ELISA plates,washed and bound antibodies were detected using secondary antibodies andOPD. Binding in the presence of the listed liposomes is shown andcompared to 3G4 antibody binding in the absence of any liposome.

[0230]FIG. 21. Binding of chimeric 3G4 to phospholipids. The chimeric3G4 antibody (ch3G4) was prepared as described in Example XIX.Phospholipids (PS, PI, PE, PC, SM, CL, PG and PA) were adsorbed toplastic of microtiter plates. After blocking, chimeric 3G4 antibody wasadded at the concentrations shown. The plates were washed and the boundchimeric 3G4 antibody was detected via secondary antibody binding anddevelopment.

[0231]FIG. 22. Localization of chimeric 3G4 to tumor vascularendothelium in vivo. Biotinylated ch3G4 (top panels) and control IgG(bottom panels) were administered to mice bearing MD-MBA-435s tumors.Tumor sections were stained with Cy3-conjugated streptavidin to detectthe biotinylated antibodies (left panels). Staining with the MECA 32antibody followed by FITC-tagged anti-rat IgG secondary antibody wasconducted to detect vascular endothelium (middle panels). The red andgreen images are merged (right panels), whereupon biotinylated proteinsbound to the tumor vascular endothelium appear yellow. The coincidentstaining of the localized 3G4 antibody and the MECA 32 marker of thevascular endothelium is shown by the yellow color on the superimposedimages (top right).

[0232]FIG. 23. Enhancement of macrophage phagocytosis of PS-positivecells by 3G4. HL-60 tumor cells were labeled with the green fluorescentdye CFDA, and PS exposure was induced by 200 μM H₂O₂. Treated cells wereharvested and opsonized for 1 hr using 5 μg/ml 3G4 or an isotype-matchedcontrol antibody (BBG3). Target cells were then added to macrophages,which were isolated from mouse bone marrow and cultured in chamberslides for 5 days in media containing 5 ng/ml GM-CSF. After 2 hrs, theslides were fixed and phagocytosis was visually counted under thefluorescent microscope. Results are presented as the percentage ofphagocytosing macrophages (macrophages that have phagocytosed at leastone tumor cell).

[0233]FIG. 24A and FIG. 24B. Induction of PS exposure on endothelialcells by docetaxel. Human umbilical vein endothelial cells (HUVEC) andhuman microvessel endothelial cells (HMVEC) were treated with 10 nM ofdocetaxel for 24 hrs. Cells were harvested, washed with PBS andincubated with 3G4 at 10 μg/ml for 30 mins. on ice. The cells were thenwashed twice, FITC labeled goat anti-mouse IgG was added and the cellsincubated for a further 30 mins. on ice. The cells were then washed andanalyzed by FACS using a FACSCalibur cytometer (Becton-Dickinson, SanJose, Calif.) with CellQuest acquisition software. Both treated HUVEC(FIG. 24A) and HMVEC (FIG. 24B) show significant increases in 3G4binding as compared to untreated cells.

[0234]FIG. 25A, FIG. 25B and FIG. 25C. Induction of PS exposure on tumorcell lines by docetaxel. Mouse lewis lung carcinoma 3LL, mouse coloncarcinoma Colo26 and human breast cancer MDA-MB-435 cells were treatedwith 10 nM of docetaxel for 24 hrs. Cells were harvested, washed withPBS and incubated with 3G4 at 10 μg/ml for 30 mins. on ice. The cellswere then washed twice, FITC labeled goat anti-mouse IgG was added andthe cells incubated for a further 30 mins. on ice. The cells were thenwashed and analyzed by FACS using a FACSCalibur cytometer(Becton-Dickinson, San Jose, Calif.) with CellQuest acquisitionsoftware. The treated 3LL (FIG. 25A), Colo26 (FIG. 25B) and MDA-MB-435cells (FIG. 25C) show significant increases in 3G4 binding as comparedto untreated cells.

[0235]FIG. 26. Induction of PS exposure on human breast cancerMDA-MB-231 cells by docetaxel. Human breast cancer MDA-MB-231 cells weretreated with 10 nM of docetaxel for 24 hrs. Cells were harvested, washedwith PBS and incubated with chimeric 3G4 (ch3G4) or control, human IgGfor 30 mins. on ice. The cells were then washed twice, FITC labeledanti-IgG was added and the cells analyzed by FACS, as above. There is asignificant increase in ch3G4 binding as compared to control, human IgG.

[0236]FIG. 27. Treatment with anti-PS antibodies increases survival ofmCMV-infected mice. Balb/C mice were infected with mCMV and treated with3G4 or ch3G4 as described in Example XXI. The mice were monitored forsurvival past 90 days after infection.

[0237]FIG. 28. Treatment with the duramycin-biotin derivative, DLBincreases survival of mCMV-infected mice. Balb/C mice were infected withmCMV and treated with DLB as described in Example XXII. The mice weremonitored for survival past 90 days after infection.

[0238]FIG. 29A and FIG. 29B. Binding of chimeric 3G4 to cells infectedwith Vaccinia virus. U937 cells were infected with Vaccinia virus andstained with the chimeric 3G4 antibody (ch3G4) or control human IgG(HIgG) on day 2 after infection. FIG. 29A, uninfected U-937 cells. FIG.29B, Vaccinia virus-infected U937 cells. The peaks in FIG. 29A and FIG.29B are: left (red) peak, secondary antibody alone control; middle(blue) peak, control HIgG; right (green) peak, ch3G4.

[0239]FIG. 30A, FIG. 30B, FIG. 30C and FIG. 30D. Inhibition of Pichindevirus replication in vitro by 3G4 antibody. Vero cells were infectedwith Pichinde virus at an m.o.i. of 0.01 pfu/cell. The infected cellswere treated with 100 μg/ml of 3G4 (FIG. 30A) or isotype-matched controlantibody, GV39G (FIG. 30B). On day 2 after infection, the cells wereharvested with trypsin and allowed to adhere to slides. The cells werefixed with acetone, and stained with anti-PIC rabbit polyclonal serumfollowed by goat anti-rabbit biotin conjugated secondary antibody.Infected cells are stained red-brown. Secondary antibody alone producedno staining (FIG. 30C). The % infected cells in the 3G4 vs. controltreated cells is also shown (FIG. 30D).

[0240]FIG. 31. Duramycin-Human IgG (HIgG) conjugate inhibits MethA tumorgrowth in vivo. BALB/c mice bearing MethA tumor cells were treated withthe duramycin-HIgG conjugate (D-SIAB)_(n)HIgG, in which duramycin isconjugated to HIgG using the SIAB linker, or with control HIgG asdescribed in Example XXV.

[0241]FIG. 32. Duramycin conjugate is not cytotoxic. The naturallyoccurring duramycin compound and the biotinylated duramycin construct,DLB were tested for cytotoxic effects on human umbilical veinendothelial cells (HUVEC) using an MTT assay.

[0242]FIG. 33. Duramycin-antibody conjugate enhances macrophagephagocytosis of apoptotic cells. A duramycin-antibody conjugate wasconstructed by linking duramycin to C44, a mouse IgG_(2a) antibody, tocreate duramycin-C44 (DuC44). Apoptotic HL-60 cells were incubated withmouse bone-marrow derived macrophages in the presence of DuC44, acontrol mouse antibody, BBG3 and the 3G4 antibody. Phagocytosis wasevaluated as percent phagocytes positive for uptake. Data are meanvalues±S.E.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0243] Solid tumors and carcinomas account for more than 90% of allcancers in man. Although the use of monoclonal antibodies andimmunotoxins has been investigated in the therapy of lymphomas andleukemias (Vitetta et al, 1991), these agents have been disappointinglyineffective in clinical trials against carcinomas and other solid tumors(Abrams and Oldham, 1985). A principal reason for the ineffectiveness ofantibody-based treatments is that macromolecules are not readilytransported into solid tumors. Even once within a tumor mass, thesemolecules fail to distribute evenly due to the presence of tightjunctions between tumor cells, fibrous stroma, interstitial pressuregradients and binding site barriers (Denekamp, 1990; Dvorak et al.,1991).

[0244] In developing new strategies for treating solid tumors, themethods that involve targeting the vasculature of the tumor, rather thanthe tumor cells, offer distinct advantages. An effective destruction orblockade of the tumor vessels arrests blood flow through the tumor,resulting in an avalanche of tumor cell death. Antibody-toxin andantibody-coagulant constructs, examples of VTA which selectively destroyand/or occlude tumor blood vessels, have already been used to greateffect in the specific targeting and destruction of tumor vasculature,resulting in tumor necrosis (Burrows et al., 1992; Burrows and Thorpe,1993; WO 93/17715; WO 96/01653; Huang et al., 1997; each incorporatedherein by reference).

[0245] VTAs exert their primary action on the pre-existing blood vesselsof solid tumors, and differ from anti-angiogenic agents that prevent newblood vessel formation. There are numerous advantages of VTAs over othercancer therapies. First, a single vessel provides the nutrition for andfacilitates removal of waste products of metabolism from hundreds orthousands of tumor cells, and only has to be damaged at one point toblock blood flow upstream and downstream. VTAs are thus particularlyeffective on established tumors. Second, endothelial cell killing,although one useful mechanism, is not required. A change of shape orlocal initiation of blood coagulation can be sufficient. Third, theendothelial cell is adjacent to the blood stream, ensuring adequate drugdelivery. Fourth, the target is a normal diploid cell that is unlikelyto acquire genetic mutations that render it drug resistant. Fifth, asurrogate marker of biological activity, i.e., blood flow, ismeasurable.

[0246] Sixth, temporary effects on vascular function may be sufficientfor significant anti-tumor effects. Studies indicate that over 99% oftumor cells in vivo can be killed during a 2 hour period of ischemia.Finally, unlike angiogenesis inhibitors, VTAs only require intermittentadministration to synergize with conventional treatments, rather thanchronic administration over months or years.

[0247] Cytotoxic VTAs are described in the following patents: U.S. Pat.Nos. 5,660,827, 5,776,427, 5,855,866, 5,863,538, 5,965,132, 6,004,554,6,051,230, 6,261,535 and 6,451,312, each incorporated herein byreference. Where antibodies, growth factors or other binding ligands areused to specifically deliver a coagulant to the tumor vasculature, suchagents are termed “coaguligands”. Coaguligand VTAs are described in thefollowing patents: U.S. Pat. Nos. 6,093,399, 6,004,555, 5,877,289 and6,036,955, each incorporated herein by reference.

[0248] A currently preferred coagulant for use in coaguligands istruncated Tissue Factor (tTF) (Huang et al., 1997; WO 96/01653; U.S.Pat. No. 5,877,289). TF is the major initiator of blood coagulation (Rufet al., 1991; Edgington et al., 1991). At sites of injury, FactorVII/VIIa in the blood comes into contact with, and binds to, TF on cellsin the perivascular tissues. The TF:VIIa complex, in the presence of thephospholipid surface, activates factors IX and X. This, in turn, leadsto the formation of thrombin and fibrin and, ultimately, a blood clot(Ruf and Edgington, 1994).

[0249] The recombinant, truncated form of tissue factor (tTF), lackingthe cytosolic and transmembrane domains, is a soluble protein that hasabout five orders of magnitude lower coagulation inducing ability thannative TF (Stone et al., 1995; Huang et al., 1997). This is because TFneeds to be associated with phospholipids for the complex with VIIa toactivate IXa or Xa efficiently. However, when tTF is delivered to tumorvascular endothelium by means of a targeting antibody or agent, it isbrought back into proximity to a lipid surface and regains thrombogenicactivity. (Huang et al., 1997; U.S. Pat. Nos. 6,093,399, 6,004,555,5,877,289 and 6,036,955). A coaguligand is thus created that selectivelythromboses tumor vasculature.

[0250] Truncated TF has several advantages that commend its use invascular targeted coaguligands: human tTF is readily available, and thehuman protein will have negligible or low immunogenicity in man; humantTF is fully functional in experimental animals, including mice; andtargeted tTF is highly potent because it triggers the activation of acascade of coagulation proteins, giving a greatly amplified effect (U.S.Pat. Nos. 6,093,399, 6,004,555, 5,877,289 and 6,036,955).

[0251] A range of suitable target molecules that are available on tumorendothelium, but largely absent from normal endothelium, have beendescribed. For example, expressed targets may be utilized, such asendoglin, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a TIE, a ligandreactive with LAM-1, a VEGF/VPF receptor, an FGF receptor, α_(v)β₃integrin, pleiotropin or endosialin (U.S. Pat. Nos. 5,855,866 5,877,289;Burrows et al., 1992; Burrows and Thorpe, 1993; Huang et al., 1997; Liuet al., 1997; Ohizumi et al., 1997; each incorporated herein byreference).

[0252] Adsorbed targets are another suitable group, such as VEGF, FGF,TGFβ, HGF, PF4, PDGF, TIMP, a ligand that binds to a TIE or atumor-associated fibronectin isoform (U.S. Pat. Nos. 5,877,289,5,965,132, 6,051,230 and 6,004,555). Fibronectin isoforms are ligandsthat bind to the integrin family of receptors. Tumor-associatedfibronectin isoforms are targetable components of both tumor vasculatureand tumor stroma. The monoclonal antibody BC-1 (Carnemolla et al., 1989)specifically binds to tumor-associated fibronectin isoforms.

[0253] Other targets inducible by the natural tumor environment orfollowing intervention by man are also targetable entities, as describedin U.S. Pat. Nos. 5,776,427, 5,863,538 and 6,036,955. When used inconjunction with prior suppression in normal tissues and tumor vascularinduction, MHC Class II antigens may also be employed as targets (U.S.Pat. Nos. 5,776,427, 5,863,538, 6,004,554 and 6,036,955).

[0254] One currently preferred target for clinical applications isvascular endothelial adhesion molecule-1 (VCAM-1) (U.S. Pat. Nos.5,855,866, 5,877,289, 6,051,230, 6,004,555 and 6,093,399). VCAM-1 is acell adhesion molecule that is induced by inflammatory cytokines IL-1α,IL-4 (Thornhill et al., 1990) and TNFα (Munro, 1993) and whose role invivo is to recruit leukocytes to sites of acute inflammation(Bevilacqua, 1993).

[0255] VCAM-1 is present on vascular endothelial cells in a number ofhuman malignant tumors including neuroblastoma (Patey et al., 1996),renal carcinoma (Droz et al., 1994), non-small lung carcinoma (Staal-vanden Brekel et al., 1996), Hodgkin's disease (Patey et al., 1996), andangiosarcoma (Kuzu et al., 1993), as well as in benign tumors, such asangioma (Patey et al., 1996) and hemangioma (Kuzu et al., 1993).Constitutive expression of VCAM-1 in man is confined to a few vessels inthe thyroid, thymus and kidney (Kuzu et al., 1993; Bruijn and Dinklo,1993), and in the mouse to vessels in the heart and lung (Fries et al.,1993).

[0256] Certain of the data presented herein even further supplementthose provided in U.S. Pat. Nos. 5,855,866, 5,877,289, 6,051,230,6,004,555 and 6,093,399, and show the selective induction of thrombosisand tumor infarction resulting from administration of an anti-VCAM-1•tTFcoaguligand. The results presented were generated using mice bearingL540 human Hodgkin lymphoma. When grown as a xenograft in SCID mice,this tumor shows close similarity to the human disease with respect toexpression of inflammatory cytokines (Diehl et al., 1985) and thepresence of VCAM-1 and other endothelial cell activation molecules onits vasculature.

[0257] Using a covalently-linked anti-VCAM-1•tTF coaguligand, in whichtTF was directly linked to the anti-VCAM-1 antibody, it is shown hereinthat the coaguligand localizes selectively to tumor vessels, inducesthrombosis of those vessels, causes necrosis to develop throughout thetumor and retards tumor growth in mice bearing solid L540 Hodgkintumors. Tumors generally needed to be at least about 0.3 cm in diameterto respond to the coaguligand, because VCAM-1 was absent from smallertumors. Presumably, in small tumors, the levels of cytokines secreted bytumor cells or host cells that infiltrate the tumor are too low forVCAM-1 induction. This is in accordance with the studies in U.S. Pat.Nos. 5,855,866, 5,877,289, 6,051,230, 6,004,555 and 6,093,399, where theinventions were shown to be most useful in larger solid tumors.

[0258] Although VCAM-1 staining was initially observed more in theperiphery of the tumor, the coaguligand evidently bound to and occludedblood transporting vessels—as it was capable of curtailing blood flow inall tumor regions. Furthermore, one of the inventors contemplates thatthe thrombin generation caused by the initial administration of thecoaguligand likely leads to further VCAM-1 induction on central vessels(Sluiter et al., 1993), resulting in an amplified signal and evidentdestruction of the intratumoral region. This type of coagulant-inducedexpression of further targetable markers, and hence signalamplification, is also disclosed in U.S. Pat. No. 6,036,955.

[0259] As shown herein, although localization to VCAM-1-expressingvessels in the heart and lungs of mice was observed upon administrationof an anti-VCAM-1 coaguligand, this construct did not induce thrombosisin such non-tumor sites. Furthermore, the anti-VCAM-1 coaguligand was nomore toxic to mice than was a control coaguligand of irrelevantspecificity, again indicating that the constitutive expression of VCAM-1on heart and lung vessels did not lead to toxicity. This data isimportant to the immediate clinical progress of coaguligand therapy,given that VCAM-1 is a naturally occurring marker of tumor vascularendothelium in humans. However, this phenomenon also provided theinventors with a unique insight, leading to a totally different approachto tumor vasculature destruction.

[0260] A. Tumor Treatment with Naked Antibodies to Aminophospholipids

[0261] The inventors sought to understand the mechanism behind theability of the anti-VCAM-1 coaguligand to bind to the VCAM-1constitutively expressed on blood vessels in the heart and lungs, andyet not to cause thrombosis in those vessels. There are numerousscientific possibilities for this empirical observation, generallyconnected with the prothrombotic nature of the tumor environment and anyfibrinolytic predisposition in the heart and lungs.

[0262] Generally, there is a biological equilibrium between thecoagulation system (fibrin deposition) and the fibrinolytic system(degradation of fibrin by enzymes). However, in malignant disease,particularly carcinomas, this equilibrium is disrupted, resulting in theabnormal activation of coagulation (hypercoagulability or the“prothrombotic state”). Despite extensive research, a clear molecularexplanation for the prothrombotic nature of the tumor environment couldnot be discerned until recently.

[0263] After detailed analyses of many possible options, the inventorsreasoned that the failure of the anti-VCAM-1 coaguligand to causethrombosis in vessels of normal tissues was due to the absence of theaminophospholipid, phosphatidylserine (PS) from the luminal surface ofsuch vessels. To complete the theory, therefore, not only wouldphosphatidylserine have to be shown to be absent from these normalvessels, but its presence on the luminal side of tumor-associatedvessels would have to be demonstrated.

[0264] The inventors therefore used immunohistochemical staining toevaluate the distribution of a monoclonal anti-phosphatidylserine(anti-PS) antibody injected intravenously into tumor-bearing mice. Thesestudies revealed that the VCAM-1 expressing vessels in the heart andlungs lacked PS, whereas the VCAM-1 expressing vessels in the tumorexpressed PS. The need for surface PS expression in coaguligand actionis further indicated by the inventors' finding that annexin V, whichbinds to PS, blocks anti-VCAM-1-tTF coaguligand action, both in vitroand in vivo.

[0265] The lack of thrombotic effect of the anti-VCAM-1 coaguligand onnormal heart and lung vessels was thus explained, at least in part: theabsence of the aminophospholipid, phosphatidylserine, means that thenormal vessels lack a procoagulant surface upon which coagulationcomplexes can assemble. In the absence of surface PS, anti-VCAM-1•tTFbinds to VCAM-1 expressing heart and lung vessels, but cannot inducethrombosis. In contrast, VCAM-1 expressing vessels in the tumor showcoincident expression of surface PS. The coaguligand thus binds to tumorvessels and activates coagulation factors locally to form an occlusivethrombus.

[0266] In addition to delineating the tumor-specific thrombotic effectsof anti-VCAM-1 coaguligands, the specific expression of theaminophospholipid, phosphatidylserine, on the luminal surface of tumorblood vessels also allowed the inventors to explain the prothromboticphenotype observed, but not understood, in earlier studies. The PSexpression plays a significant role in the prothrombotic state of tumorvasculature.

[0267] Following their discovery that the representativeaminophospholipid, phosphatidylserine, was specifically expressed on theluminal surface of tumor blood vessels, but not in normal blood vessels,the inventors reasoned that other aminophospholipids had potential astargets for therapeutic intervention. The inventors therefore developedtumor vasculature targeting and treatment methods based on targeting theaminophospholipids phosphatidylserine and phosphatidylethanolamine (PE).

[0268] A particularly surprising aspect of the inventors' studies wasthat administration of an unconjugated anti-aminophospholipid antibodywas effective in tumor treatment. This gave rise to important newavenues of tumor treatment using unconjugated or “naked” antibodies thatbind to aminophospholipids. These tumor vasculature targeting andtreatment methods are described in U.S. Pat. No. 6,406,693, incorporatedherein by reference. Although anti-tumor effects in art-accepted animalmodels are demonstrated in U.S. Pat. No. 6,406,693, and extended herein,the ability of aminophospholipids to act as safe and effectivetargetable markers of tumor vasculature could not have been predictedfrom studies previous to U.S. Pat. No. 6,406,693.

[0269] Once the discovery of aminophospholipids as specific markers oftumor vasculature had been proven, the inventors began to develop arange of aminophospholipid-targeted immunotoxins and coaguligands foruse in tumor treatment. As explained in U.S. Pat. No. 6,406,693, thisled to the unexpected discovery of naked anti-aminophospholipidantibodies for use in tumor treatment. In investigating the potential ofaminophospholipid targeting in the context of delivering a toxin orcoagulant to the tumor vasculature, the inventors serendipitously foundthat naked anti-PS antibodies had a destructive effect on tumorvasculature in vivo in the absence of any additional effector moiety.The ability of anti-aminophospholipid antibodies to both specificallylocalize to tumor vasculature and to exert a concomitant destructiveeffect, leading to tumor necrosis, was most unexpected.

[0270] The present invention provides surprising and improved, “secondgeneration” anti-PS antibodies for use, amongst other embodiments, asnaked antibodies in tumor treatment. A panel of second generationanti-PS antibodies is disclosed herein, of which the monoclonalantibodies 9D2 and 3G4 (ATCC 4545) are currently preferred, along withparticular immunization and screening techniques for the generation andselection of further antibodies with such advantageous properties. It isalso shown herein that vascular damage to tumor vessels by anti-PSantibodies is mediated, at least in part, through host effectors. Theseand other insights of the present inventors allow for naked antibodytreatment to be optimized, both when used alone, and in combination withother anti-cancer agents, as taught herein.

[0271] B. Tumor Treatment Using Antibodies to Anionic Phospholipids

[0272] U.S. Pat. No. 6,406,693 explains that the aminophospholipidsphosphatidylserine and phosphatidylethanolamine are normally segregatedto the inner surface of the plasma membrane bilayer in different cells(Gaffet et al., 1995; Julien et al., 1995) and that this lipidsegregation creates an asymmetric transbilayer. Although the existenceof membrane asymmetry has been discussed for some time, the reason forits existence and the mechanisms for its generation and control arepoorly understood (Williamson and Schlegel, 1994), particularly in cellsother than platelets.

[0273] The inventors earlier demonstrated that PS is translocated to thesurface of tumor vascular endothelial cells and that this occurs, atleast in significant part, independently of apoptotic or othercell-death mechanisms (U.S. Pat. No. 6,406,693). Thus, PS surfaceexpression in the tumor environment is not a consequence of cell death,nor does it trigger immediate cell destruction. Despite PS exposurebeing detected consistently on intact vascular endothelial cells invarious solid tumors, the tumor vascular endothelium is not franklyapoptotic, but is morphologically sound (although different to that innormal tissues) and metabolically active. This is important fortherapeutic methods based on PS targeting, meaning that PS translocationto the outer membrane in tumor vascular endothelial cells issufficiently stable for PS to serve as a targetable entity forsuccessful therapy (using either naked antibodies or therapeuticconjugates).

[0274] Despite the important discoveries of U.S. Pat. No. 6,406,693 (andU.S. Pat. No. 6,312,694, see below), the suggestions forphospholipid-based targeting of tumor vascular endothelial cells wereconfined to the targeting of aminophospholipids, such as PS and PE.Through the development of biological tools with exquisite specificityfor different phospholipids and aminophospholipids, the presentinventors have now identified a new category of phospholipids that aresurprisingly upregulated on tumor vascular endothelial cells. These arethe anionic phospholipids, which are shown herein to also be specificand stable markers of tumor vasculature, permitting therapeuticintervention using both naked antibodies and immunoconjugates that bindto anionic phospholipids.

[0275] Anionic phospholipids are largely absent from the surface ofresting mammalian cells under normal conditions. Phosphatidylserine,which is the most abundant anionic phospholipid of the plasma membrane,is tightly segregated to the internal leaflet of the plasma membrane inmost cell types under normal conditions (Williamson and Schlegel, 1994;Zwaal and Schroit, 1997). Phosphatidylinositol (PI), another majoranionic phospholipid, is also predominantly situated in the internalleaflet of the plasma membrane (Calderon and DeVries, 1997). The minoranionic phospholipids, phosphatidic acid (PA) and phosphatidylglycerol(PG), have only been examined in a few cells types, but they also appearto be mainly situated in the internal leaflet of the plasma membrane(Hinkovska-Galcheva et al., 1989). Cardiolipin (CL), another anionicphospholipid, is present in the mitochondrial membrane and is absentfrom the plasma membrane (Daum, 1985).

[0276] The neutral phospholipids are also asymmetrically distributed inthe plasma membrane. The neutral aminophospholipid,phosphatidylethanolamine (PE) is predominately on the internal leaflet.The choline-containing neutral phospholipids, phosphatidylcholine (PC)and sphingomyelin (SM), are predominantly on the external leaflet.

[0277] PS asymmetry, along with that of PE, is maintained by anATP-dependent transporter, aminophospholipid translocase (Mg²⁺ ATPase),which catalyzes the transport of aminophospholipids from the externalleaflet to the internal leaflet of the plasma membrane (Seigneuret andDevaux, 1984). Loss or collapse of PS and PE asymmetry results from theoutward movement of these phospholipids in the plasma membrane and iscaused either by inhibition of the translocase (Bitbol et al., 1987;Comfurius et al., 1990), activation of PS transporters and/or activationof scramblase enzymes, Ca²⁺ dependent enzymes that transport all lipidsbidirectionally (Zhao et al., 1998).

[0278] Loss of PS asymmetry is observed under different pathological andphysiological conditions, including cell injury, programmed cell deathand apoptosis (Blankenberg et al., 1998; Bombeli et al., 1997), cellaging (Herrmann and Devaux, 1990), activation of platelets (Rote et al.,1993; Zwaal et al., 1989), injury (Boyle et al., 1996) and malignanttransformation (Sugimura et al., 1994). Exposure of PS also plays a rolein intercellular fusion of myoblasts (Sessions and Horwitz, 1981) andtrophoblasts (Adler et al., 1995), cell migration (Vogt et al., 1996)and cell degranulation (Demo et al., 1999). Endothelial cellsexternalize PS in response to increased Ca²⁺ fluxes induced by thrombin(Qu et al., 1996), calcium ionophore or phorbol esters (Julien et al.,1997), hyperlipidemia (Lupu et al., 1993), and non-lytic concentrationsof complement proteins C5b-9 (Christiansen et al., 1997). Spontaneous PSexposure has been also observed in malignant cells in the absence ofexogenous activators or cell injury (Utsugi et al., 1991).

[0279] Several major consequences follow membrane PS exposure.Phagocytic macrophages recognize, attach and eliminate PS-positivesenescent and apoptotic cells (McEvoy et al., 1986; Tait and Smith,1999). PS also mediates attachment of T lymphocytes tothrombin-activated endothelial cells (Qu et al., 1996). The complementsystem is activated by PS and contributes to the lysis of PS-positivecells (Test and Mitsuyoshi, 1997). Finally, PS exposure contributes to aprocoagulant shift on the endothelium (Williamson and Schlegel, 1994;Bombeli et al., 1997) by providing a negatively charged lipid surfacefor assembly and activation of coagulation complexes (Bevers et al.,1985; Dachary-Prigent et al., 1996). The prothrombotic character of thetumor endothelium has long been recognized (Donati and Falanga, 2001).

[0280] Despite the focus on PS in the scientific literature, and theinventors' earlier work confined to aminophospholipids such as PS and PE(U.S. Pat. Nos. 6,406,693 and 6,312,694), the present inventorshypothesized that a wider category of phospholipids could become exposedon tumor vasculature. Due to the increased stress conditions of thetumor microenvironment, the inventors reasoned that a range of anionicphospholipids could be upregulated on tumor vasculature, providingpotential new opportunities for therapeutic intervention.

[0281] The inventors realized that injury and activation of tumorendothelium are caused by: 1) tumor-derived cytokines, such asinterleukin-1 and tumor necrosis factor, which activate the endotheliumand induce expression of cell adhesion molecules (Shaughnessy et al.,1989; Orr et al., 2000); 2) reactive oxygen species (ROS) generated byleukocytes that adhere to the endothelium (Orr et al., 2000); and 3) ROSgenerated by tumor cells themselves as a byproduct of metabolism(Shaughnessy et al., 1989; Soares et al., 1994) or as a result ofexposure to hypoxia followed by reoxygenation (Zulueta et al., 1995).These observations suggested that Ca²⁺ fluxes might be generated bythese stresses within the tumor endothelium that, in turn, causeexposure of PS and PE, through activation of scramblase or inhibition ofaminophospholipid translocase.

[0282] However, the inventors extended these insights to the hypothesisthat anionic phospholipids, not just the aminophospholipids PS and PE,would be upregulated on tumor vasculature. To detect cell surfaceanionic phospholipids, the inventors generated a new monoclonalantibody, 9D2, which reacts with anionic but not neutral phospholipids.9D2 thus differentiates from general aminophospholipid binding agents,as it binds to the anionic aminophospholipid, PS, but not to the neutralaminophospholipid, PE. The 9D2 antibody is also more specific foranionic phospholipids than is the natural ligand, annexin V, whichstrongly binds to PE, in addition to anionic phospholipids (Blankenberget al., 1998).

[0283] As detailed in the present application, the inventors found that9D2 and annexin V localize specifically to tumor endothelium afterintravenous injection to mice bearing various types of solid tumors.This finding validates the inventors' hypothesis that anionicphospholipids routinely become exposed on the surface of tumor vascularendothelium and can be used as target molecules for tumor therapy (andimaging). The present invention thus provides a range of new methods andantibody-based compositions for use in targeting anionic phospholipidsand treating tumors, both in terms of naked antibodies and in thedelivery of cytotoxic drugs, cytokines, coagulants and such like. Inaddition to targeting PS, as taught in U.S. Pat. Nos. 6,406,693 and6,312,694, the currently preferred anionic phospholipids for targetingby the present invention are PI, a major anionic phospholipid, PA andPG, with targeting CL also being contemplated in certain embodiments.

[0284] One of the major findings to emerge from the present invention isthat anionic phospholipids are exposed on the surface of tumorendothelium (Example VI). This phenomenon was demonstrated using twoindependent reagents that bind selectively to anionic phospholipids: amonoclonal antibody, 9D2, developed by the inventors particularly tovalidate this point, and annexin V. The 9D2 antibody and competingantibodies are further preferred components of the present invention.

[0285] 9D2 antibody and annexin V bind with high affinity andspecificity to anionic phospholipids adsorbed to plastic, as liposomes,or presented on the membrane surface of activated or apoptoticendothelial cells in vitro. 9D2 binds strongly to PS, PA and CL, butmore weakly to PI and PG. Annexin V binds to PE in addition to PS, CL,PA, PI and PG, as found previously (Andree et al., 1990; Schlaepfer etal., 1987; Boustead et al., 1993; Blackwood and Ernst, 1990).Recognition of anionic phospholipids by 9D2 antibody was identical inthe presence and absence of serum, indicating that binding does notrequire serum co-factors. Binding of 9D2 to anionic phospholipids, didnot require Ca²⁺ ions, whereas the binding of annexin V did requireCa²⁺.

[0286] Cross-blocking studies on PS-coated plates showed that 9D2 andannexin V do not block each other's binding to PS. This indicates thatthe two reagents recognize different epitopes on the PS molecule, or,more likely, differently packed forms of PS. Annexin V is thought tobind to planar PS surfaces, whereas anti-PS antibodies are thought tobind to hexagonally packed PS (Rauch and Janoff, 1990). Both forms areprobably present on PS-coated plates. These practical cross-blockingstudies (Example VI) also serve to show that antibodies whicheffectively compete for binding to anionic phospholipids, i.e., bind toessentially the same epitope, can be readily identified once a referenceantibody (e.g. 9D2) is provided.

[0287] The present application also shows that 9D2 antibody and annexinV specifically localize to tumor vessels, and to tumor cells in andaround necrotic regions of all tumors examined in vivo (Example VI).Between 15 and 40% of blood vessels in the tumors had anionicphospholipid-positive endothelium. In contrast, none of the bloodvessels in normal tissues had detectable externalized anionicphospholipids.

[0288] The specificity of staining of tumor vasculature by 9D2 wasdemonstrated by: 1) the lack of tumor vessel staining by control ratIgM; 2) the blocking of 9D2 or annexin V binding to H₂O₂-treatedendothelial cells in vitro by liposomes prepared from anionicphospholipids, but not neutral phospholipids; 3) the finding thatextraction of phospholipids from tumor sections with detergents ororganic solvents abolished staining; and 4) the lack of localization ofeither 9D2 or annexin V to the quiescent endothelium in normal organs.

[0289] The main anionic phospholipid that is localized by 9D2 or annexinV on tumor vasculature is likely to be PS, as this is the most abundantanionic phospholipid and its exposure on the cell surface is regulatedby environmental influences or injury. However, other anionicphospholipids (e.g., PI, PA, PG) are also likely to be exposed, despitebeing less abundant.

[0290] Although not detected by 9D2, the major neutral phospholipid, PE,is likely to contribute, together with PS, to the annexin localizationobserved on tumor vessels. PE is also known to be exposed on tumorendothelium, and the position of PE in the plasma membrane is regulatedin a similar manner to PS (U.S. Pat. No. 6,406,693). PE is segregated tothe internal leaflet of the plasma membrane in part by aminophospholipidtranslocase, although at a slower rate than PS (Devaux, 1992), and istransported to the external surface by scramblase (Zhou et al., 1997).PE, like PS, is also exposed during apoptosis and cell activation (Emotoet al., 1997; Umeda and Emoto, 1999).

[0291] To examine the mechanism of exposure of anionic phospholipids ontumor endothelial cells, a series of studies was performed in whichendothelial cells in vitro were treated with various factors andconditions known to be present in the tumor microenvironment (ExampleVII). Hypoxia followed by re-oxygenation, acidity, and thrombinincreased PS exposure on viable endothelial cells to between 10 and 22%of the level seen when all cells are apoptotic. Inflammatory cytokines(TNFα and IL-1) also caused a weak but definite induction of PSexposure.

[0292] These findings are consistent with the possibility that, intumors, exposure of anionic phospholipids on the vascular endothelium isinduced by hypoxia/reoxygenation in combination with inflammatorycytokines, thrombin and acidity. Although the precise mechanism does notneed to be understood to practice the present invention, ROS may begenerated by tumor cells as a bi-product of metabolism or in response tohypoxia (Zulueta et al., 1995). Cytokines released by tumor cells mayinduce leukocytes adhesion molecules on the endothelium that mediateadherence of activated macrophages, polymorphonuclear cells andplatelets to tumor endothelium and further secretion of ROS. The ROS maythen induce PS translocation through oxidation of thiol-containingtransport molecules or peroxidation of lipids (Herrmann and Devaux,1990), possibly by causing an influx of Ca²⁺ or release of Ca²⁺ fromintracellular stores (Wang and Joseph, 2000).

[0293] Exposure of PS and other anionic phospholipids in part explainsthe procoagulant status of tumor endothelium that has long beenrecognized (Donati and Falanga, 2001). The anionic phospholipids providethe surface upon which coagulation factors concentrate and assemble(Bevers et al., 1985; Dachary-Prigent et al., 1996). It also provides anattachment site for circulating macrophages (McEvoy et al., 1986), Tlymphocytes (Qu et al., 1996) and polymorphonuclear cells that assistsin leukocyte infiltration into tumors.

[0294] Antibodies and other ligands that bind to anionic phospholipidscan thus be used for the targeting, imaging and/or treatment of tumorblood vessels. Anionic phospholipids are attractive as tumor targetvessels for several reasons: they are abundant (PS is present at 3×10⁶molecules per cell); they are on the luminal surface of tumorendothelium, which is directly accessible for binding by vasculartargeting agents in the blood; they are present on a major percentage oftumor endothelial cells in diverse solid tumors; and they areessentially absent from endothelium in all normal tissues.

[0295] Vascular targeting agents employing drugs or coagulants have beenshown to be highly effective, and sometimes curative, in mice with largesolid tumors (Huang et al., 1997; Nilsson et al., 2001; U.S. Pat. Nos.5,660,827, 5,776,427, 5,855,866, 5,863,538, 5,965,132, 6,004,554,6,051,230, 6,261,535, 6,093,399, 6,004,555, 5,877,289 and 6,036,955).The present invention thus provides naked antibodies and vasculartargeting agents directed against anionic phospholipids for use intargeting tumor vasculature in the diagnosis and treatment of cancer inman.

[0296] Although a precise molecular understanding of how nakedantibodies directed against anionic phospholipids and aminophospholipidsfunction in tumor treatment is not necessary in order to practice thetreatment, the inventors have contemplated several mechanisms that mayaccount for the observed endothelial cell killing. The favoredmechanisms (particularly for the 3G4 antibody described herein) are Fcdomain-mediated immune effector functions, such as antibody-dependentcellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)and antibody mediated phagocytosis. Cell-mediated cytotoxicity,complement-mediated lysis and/or apoptosis, antibody-induced cellsignaling and/or disturbances to the cytoskeleton may also be involved.

[0297] Binding of intact antibodies against anionic phospholipids andaminophospholipids, particularly 3G4, to the vascular endothelial cellsurface means that the Fc portions of the antibodies protrude into thevessel lumen. As antibody Fc fragments activate the complement pathway,the observed cellular destruction may be a result of complement-directedlysis. Antibody binding thus activates the complement-dependentcoagulation cascade, causing multi-component complexes to assemble and,ultimately, to generate a lytic complex that permeabilizes the targetcell. “Complement-activated ADCC” may also be operating in thedestruction, in which complement binds to the antibody-coated targetcell, and in which cells, such as neutrophils, having receptors forcomplement, lyse the target cell.

[0298] As the naked or unconjugated antibodies, including the antigenbinding fragments thereof, bind to anionic phospholipids andaminophospholipids at the surface of the tumor vascular endothelialcells, they will form an antibody coating on the luminal surface. Thismay function to attract immune effector cells, such as cytotoxic T cellsand/or natural killer (NK) cells, which will then exert a cell-mediatedcytotoxic effect on the vascular endothelial cells.

[0299] Antibody binding to anionic phospholipids and aminophospholipidsmay also induce apoptosis in the tumor vascular endothelial cells.Although there are no known reports of antibody binding to PS actuallyinducing apoptosis (rather than PS being a marker resulting fromapoptosis), the inventors consider this to be another possible mechanismfor the observed anti-tumor effects.

[0300] It is also possible that antibody binding to anionicphospholipids and aminophospholipids at the surface of tumor vascularendothelial cells may cause disturbances in the cytoskeletalalorganization of the cell. As the cytoskeleton plays a role in theorganization of surface membranes, and as antibody binding may disturb(or further disturb) the membrane, binding of antibodies to anionicphospholipids and aminophospholipids may transmit changes tocytoskeletal proteins that interact with the bilayer. It is alreadyknown that the spatial organization of cytoskeletal proteins controlsmembrane stability and cell shape, and it is possible that perturbationof some cytoskeletal equilibrium may have far-reaching consequences oncell integrity.

[0301] A further mechanism of operation of the invention may be thatantibody binding to anionic phospholipids and aminophospholipids at theendothelial cell surface may initiate signal transduction by, as yet,undefined pathways. Antibody binding may also disturb known signaltransduction pathways, e.g., by altering the conformation and/orinteractions of membrane receptors, signal transduction proteins,membrane channels, and the like. Signals for cell destruction(apoptosis) may be initiated or mimicked, and/orpreservation/homeostatic signals may be inhibited.

[0302] Although of scientific interest, determining the exact nature ofthe vascular destruction achieved by the naked antibodies to anionicphospholipids and aminophospholipids is not necessary to practice thetreatment. Given that the administration of these categories ofantibodies is shown to advantageously result in specific anti-tumoreffects in vivo, the treatment can be utilized irrespective of themolecular mechanism that underlies this phenomenon. The use of nakedantibodies that bind to anionic phospholipids and aminophospholipids,thus represents an important advance in tumor therapy, providingadvantages in preparation and cost.

[0303] C. Antibodies to Anionic Phospholipids and Aminophospholipids

[0304] As the present invention identifies a new category of tumorvasculature markers, the anionic phospholipids, naked antibodies andimmunoconjugates that bind to one or more anionic phospholipids,optionally in combination with aminophospholipids, can now be used intumor diagnosis and treatment.

[0305] C1. Polyclonal Antibodies

[0306] Means for preparing and characterizing antibodies are well knownin the art (see, e.g., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988; incorporated herein by reference). To preparepolyclonal antisera an animal is immunized with a composition comprisingan immunogenic anionic phospholipid and/or aminophospholipid, includingcells treated with H₂O₂ and other agents, as taught herein, and antiseracollected from that immunized animal. A wide range of animal species canbe used for the production of antisera. Typically the animal used forproduction of anti-antisera is a rabbit, mouse, rat, hamster, guinea pigor goat. Because of the relatively large blood volume of rabbits, arabbit is a preferred choice for production of polyclonal antibodies.

[0307] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the immunogen; subcutaneous, intramuscular, intradermal,intravenous, intraperitoneal and intrasplenic. The production ofpolyclonal antibodies may be monitored by sampling blood of theimmunized animal at various points following immunization. A second,booster injection, may also be given. The process of boosting andtitering is repeated until a suitable titer is achieved. When a desiredtiter level is obtained, the immunized animal can be bled and the serumisolated and stored. The animal can also be used to generate monoclonalantibodies.

[0308] As is well known in the art, the immunogenicity of a particularcomposition can be enhanced by the use of non-specific stimulators ofthe immune response, known as adjuvants. Exemplary adjuvants includecomplete Freund's adjuvant, a non-specific stimulator of the immuneresponse containing killed Mycobacterium tuberculosis; incompleteFreund's adjuvant; and aluminum hydroxide adjuvant.

[0309] It may also be desired to boost the host immune system, as may beachieved by associating anionic phospholipids and aminophospholipidswith, or coupling to, a carrier. Exemplary carriers are keyhole limpethemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such asovalbumin, mouse serum albumin or rabbit serum albumin can also be usedas carriers.

[0310] As is also known in the art, a given composition may vary in itsimmunogenicity. However, the generation of antibodies against anionicphospholipids and aminophospholipids is not particularly difficult. Forexample, highly specific anti-phosphatidylserine antibodies were raisedin rabbits immunized by intramuscular injections ofphosphatidylserine-containing polyacrylamide gels and withphosphatidylserine-cytochrome c vesicles (Maneta-Peyret et al., 1988;1989; each incorporated herein by reference). The use of acrylamideimplants enhanced the production of antibodies (Maneta-Peyret et al.,1988; 1989). The anti-phosphatidylserine antibodies raised in thismanner are able to detect phosphatidylserine in situ on human platelets(Maneta-Peyret et al., 1988). The groups of Inoue, Rote and Rauch havealso developed anti-PS and anti-PE antibodies (see below).

[0311] Although the generation of antibodies against anionicphospholipids and aminophospholipids can be achieved by various means,certain preferred methods are described herein in Example IV.

[0312] C2. Monoclonal Antibodies

[0313] Various methods for generating monoclonal antibodies (MAbs) arealso now very well known in the art. The most standard monoclonalantibody generation techniques generally begin along the same lines asthose for preparing polyclonal antibodies (Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988; incorporated herein byreference). A polyclonal antibody response is initiated by immunizing ananimal with an immunogenic anionic phospholipid and/or aminophospholipidcomposition and, when a desired titer level is obtained, the immunizedanimal can be used to generate MAbs. Preferably, the particularscreening and selection techniques disclosed herein are used to selectantibodies with the sought after properties.

[0314] MAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with the selected immunogen composition.The immunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep and frog cells isalso possible. The use of rats may provide certain advantages (Goding,1986, pp. 60-61; incorporated herein by reference), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

[0315] Following immunization, somatic cells with the potential forproducing the desired antibodies, specifically B lymphocytes (B cells),are selected for use in the MAb generating protocol. These cells may beobtained from biopsied spleens, tonsils or lymph nodes, or from aperipheral blood sample. Spleen cells and peripheral blood cells arepreferred, the former because they are a rich source ofantibody-producing cells that are in the dividing plasmablast stage, andthe latter because peripheral blood is easily accessible. Often, a panelof animals will have been immunized and the spleen of animal with thehighest antibody titer will be removed and the spleen lymphocytesobtained by homogenizing the spleen with a syringe. Typically, a spleenfrom an immunized mouse contains approximately 5×10⁷ to 2×10⁸lymphocytes.

[0316] The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

[0317] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984; each incorporated herein by reference). For example, where theimmunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F,4B210 or one of the above listed mouse cell lines; and U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6, are all useful in connectionwith human cell fusions.

[0318] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 4:1 proportion, though the proportion may varyfrom about 20:1 to about 1:1, respectively, in the presence of an agentor agents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976; each incorporated herein by reference),and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, byGefter et al. (1977; incorporated herein by reference). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986; incorporated herein by reference).

[0319] Fusion procedures usually produce viable hybrids at lowfrequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0320] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannotsurvive. The B cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B cells.

[0321] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired reactivity. The assay shouldbe sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

[0322] The selected hybridomas would then be serially diluted and clonedinto individual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide MAbs in high concentration. The individualcell lines could also be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations.

[0323] MAbs produced by either means will generally be further purified,e.g., using filtration, centrifugation and various chromatographicmethods, such as HPLC or affinity chromatography, all of whichpurification techniques are well known to those of skill in the art.These purification techniques each involve fractionation to separate thedesired antibody from other components of a mixture. Analytical methodsparticularly suited to the preparation of antibodies include, forexample, protein A-Sepharose and/or protein G-Sepharose chromatography.

[0324] D. Second Generation Antibodies to Anionic Phospholipids andAminophospholipids

[0325] The present invention provides “second generation” antibodiesthat bind to aminophospholipids and anionic phospholipids, whichantibodies have improved properties and/or do not suffer from thedrawback associated with the antibodies in the prior art. A panel ofsuch antibodies is disclosed herein, of which the monoclonal antibodies9D2 and 3G4 are currently preferred, with the 3G4 (ATCC 4545) antibodybeing particularly preferred. The invention also provides particularimmunization and screening techniques, which permit “like” or“competing” antibodies with advantageous properties and/or lessdrawbacks to be produced.

[0326] D1. Antibody Properties

[0327] The second generation antibodies of the invention bind toaminophospholipids and anionic phospholipids and yet do not havepathogenic properties usually associated with antibodies to suchphospholipids. This was made possible, in part, by the new immunizationand screening techniques developed by the inventors.

[0328] Anti-phospholipid syndrome(s) (APS) are associated withautoantibodies termed “anti-cardiolipin” antibodies and “lupusanticoagulant antibodies”. These syndromes are associated with apredisposition towards venous and arterial thromboemboli,thrombocytopenia and a number of neurological syndromes. Theanti-phospholipid antibodies in these patients are thus “pathogenicantibodies”.

[0329] Although described for years as “anti-phospholipid antibodies”and “anti-PS antibodies”, such pathogenic antibodies in fact recognizeprotein cofactors that bind to cardiolipin, PS or both, not thephospholipids themselves (Galli et al., 1990; 1993; McNeil et al., 1990;Rote, 1996). Anti-cardiolipin antibodies recognize a particular region(between residue 281 and residue 288) on β2-glycoprotein I, whereaslupus anticoagulant antibodies recognize prothrombin. Similarly, anti-PEantibodies that occur in disease states bind to PE in combination withproteins, such as low and high molecular weight kininogen (HK),prekallikrein and factor XI (Sugi and McIntyre, 1995; 1996a; 1996b).Based upon this type of protein recognition, the anti-phospholipidantibodies in patients displace the protein cofactors from thephospholipids, thus creating the symptoms of disease.

[0330] The antibodies of the present invention have been particularlyselected on the basis of not binding to aminophospholipids and anionicphospholipids in combination with protein cofactors, but rather are“true” anti-phospholipid antibodies. As such, the antibodies of theinvention do not bind or displace the protein cofactors from thephospholipids and are therefore safe for administration. Indeed, micetreated with the antibodies of the invention at high doses for prolongedperiods showed no changes in coagulation capability, yet mice respondwith APS when injected with anticardiolipin or lupus anticoagulantantibodies.

[0331] Irrespective of the underlying mechanisms, anti-phospholipidantibodies occurring in the human population are correlated withautoimmune diseases, e.g., systemic lupus erythematosus (Branch et al.,1987; Staub et al., 1989; Drouvalakis and Buchanan, 1998; Smimov et al.,1995; Rauch et al., 1986; Rauch and Janoff, 1990) and recurrentpregnancy loss (Rote et al., 1995; Rote, 1996; Vogt et al., 1996; 1997;Katsuragawa et al., 1997). No such symptoms have been associated whenthe antibodies of the present invention are administered to mice ormonkeys.

[0332] Also, the epitope recognized by the antibodies of the invention,such as the 9D2 and 3G4 (ATCC 4545) antibodies, is not the same as thatrecognized by annexin V. This is shown herein, as the agents do notcrossblock each others' binding to phospholipids. The epitope recognizedby the 3G4 and 9D2 antibodies is probably a hexagonally packed form ofPS, which is the immunogenic form. Annexin V likely binds to planar PSin addition to the hexagonal form. The hexagonal form of PS concentratesinto protuberances in the plasma membrane associated with cellactivation and into “blebs” on apoptotic cells. The restricteddistribution of the antibodies of the invention, such as the 9D2 and 3G4(ATCC 4545) antibodies, thus further contributes to the lack ofdetectable toxicity and lack of effect on coagulation of the antibodies.

[0333] In order to generate antibodies to aminophospholipids and anionicphospholipids with advantageous properties and/or reduced or essentiallyno side effects, the present invention provides preferred immunizationand screening methods. Other immunization techniques and antibodies havebeen reported in the literature (Umeda et al., 1989; Igarashi et al.,1991; Rote et al., 1993), including those with reported specificity forthe type of fatty acid chains involved (Levy et al., 1990; Qamar et al.,1990). However, the present immunization techniques, and particularlythe selection of antibodies that are not serum dependent, providesparticular benefits.

[0334] Umeda et al. (1989) reported the production of monoclonalantibodies recognizing stereo-specific epitopes of phosphatidylserine.However, the Umeda system suffers from the drawback of using directimmunization of phosphatidylserine into mouse spleen using aSalmonella-coated aminophospholipid sample (Umeda et al., 1989). Many ofthe antibodies reported by Umeda et al. (1989) also exhibitanticoagulant activity, which is a drawback not associated with theantibodies of the present invention. The binding profile of the 3G4antibody is different to that of the PSC8 antibody of Umeda et al.(1989).

[0335] The antibodies of the invention also have the advantage ofrecognizing all or most anionic phospholipids, which can provide moretargets for binding. Therefore, the second generation antibodies of theinvention can be defined as having substantially the same, or the same,phospholipid specificity as the 9D2 or 3G4 (ATCC 4545) antibodies, asdisclosed herein in Table 4, and as not being serum dependent.

[0336] Igarashi et al. (1991) also reported the induction of anti-PSantibodies, but again used intrasplenic immunization and only a slightincrease of the titer was observed when the antigen was again injectedintravenously. Most of the MAbs from Igarashi et al. (1991)cross-reacted with DNA and many exhibited lupus anticoagulant activity,neither of which drawbacks exist in the antibodies developed by thepresent inventors. The binding profile of the preferred, 3G4 antibody ofthe invention is also different to those of the antibodies in Table 1 ofIgarashi et al. (1991).

[0337] Others have reported the lupus anticoagulant activities of murinemonoclonal antibodies that cross react with more than one anionicphospholipid (Alving et al., 1987; Rauch and Janoff, 1990), but thepresent inventors have experienced no difficulty in obtaining antibodiesfree from lupus anticoagulant activity. This represents a distinctadvantage of the methods, antibodies and competing antibodies inaccordance with the present invention.

[0338] In addition to avoiding the use of antibodies from patients, suchas described in Rauch et al. (1986), Hasegawa et al. (1994), Ravirajanet al. (1995) and Menon et al. (1997), the present application alsodemonstrates the advantageous properties of the antibodies provided bythis invention in side-by-side comparisons with existing antibodies fromthe literature, such as the 3SB antibody described by Rote et al.(1993). Although the 3SB antibody has properties suitable for use invarious of the methods disclosed herein, the antibodies developed by thepresent inventors nonetheless out-perform the 3SB antibody incomparative studies, e.g., as shown herein by the increased anti-viraleffects of the 3G4 antibodies as opposed to the 3SB antibody (ExampleXIII).

[0339] The antibodies of the present invention can also be characterizedby their affinity. Prior to the invention, the antibodies in theliterature had relatively weak affinity (where reported). In certainembodiments, the second generation antibodies of the invention aretherefore defined as those that have an affinity for PS of at leastequal to the affinity of the 9D2 or 3G4 (ATCC 4545) antibodies for PS,in particular, the affinity when measured in an ELISA as describedherein, as disclosed in Table 3, and as not being serum dependent.

[0340] More preferably, the second generation antibodies of theinvention are defined as those having an affinity for PS of at leastequal to the affinity of the 9D2 or 3G4 (ATCC 4545) antibodies for PS,as disclosed in Table 3, and as having substantially the same, or thesame, phospholipid specificity as the 9D2 or 3G4 (ATCC 4545) antibodies,as disclosed in Table 4, and as not being serum dependent. Mostpreferably, the second generation antibodies are those having anaffinity for PS of at least equal to the affinity of the 3G4 (ATCC 4545)antibody for PS, as disclosed in Table 3, and as having the samephospholipid specificity as the 3G4 (ATCC 4545) antibody, as disclosedin Table 4, and as not being serum dependent.

[0341] D2. CDR Technologies

[0342] Antibodies are comprised of variable and constant regions. Theterm “variable”, as used herein in reference to antibodies, means thatcertain portions of the variable domains differ extensively in sequenceamong antibodies, and are used in the binding and specificity of eachparticular antibody to its particular antigen. However, the variabilityis not evenly distributed throughout the variable domains of antibodies.It is concentrated in three segments termed “hypervariable regions”,both in the light chain and the heavy chain variable domains (other thancamelized antibodies discussed below).

[0343] The more highly conserved portions of variable domains are calledthe framework region (FR). The variable domains of native heavy andlight chains each comprise four FRs (FR1, FR2, FR3 and FR4,respectively), largely adopting a β-sheet configuration, connected bythree hypervariable regions, which form loops connecting, and in somecases, forming part of, the β-sheet structure.

[0344] The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (Kabat et al., 1991, specifically incorporated herein byreference). The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.

[0345] The term “hypervariable region”, as used herein, refers to theamino acid residues of an antibody that are responsible forantigen-binding. The hypervariable region comprises amino acid residuesfrom a “complementarity determining region” or “CDR” (i.e. residues24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domainand 31-35 (H1), 50-56 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al., 1991, specifically incorporated herein byreference) and/or those residues from a “hypervariable loop” (i.e.residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain). “Framework” or “FR” residues are those variabledomain residues other than the hypervariable region residues as hereindefined.

[0346] The DNA and deduced amino acid sequences of the Vh and Vκ chainsof the 3G4 antibody (ATCC 4545) are provided herein as SEQ ID NO:1, 2, 3and 4, respectively. These sequences encompass CDR1-3 of the variableregions of the heavy and light chains of the antibody. In light of thesequence and other information provided herein, and the knowledge in theart, a range of 3G4-like and improved antibodies and antigen bindingregions can now be prepared and are thus encompassed by the presentinvention.

[0347] In certain embodiments, the invention provides at least one CDRof the antibody produced by the hybridoma deposited as ATCC 4545. Inother embodiments, the invention provides a CDR, antibody, or antigenbinding region thereof, which binds to at least a firstaminophospholipid or anionic phospholipid, preferably PS, and whichcomprises at least one CDR of the antibody produced by the hybridomadeposited as ATCC 4545.

[0348] Further aspects of the invention concern at least one CDR thathas the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or a variantor mutagenized form thereof. Other aspects of the invention concern aCDR, antibody, or antigen binding region thereof, which binds to atleast a first aminophospholipid or anionic phospholipid, preferably PS,and which comprises at least one CDR with the amino acid sequence of SEQID NO:2 or SEQ ID NO:4, or a variant or mutagenized form thereof,wherein such a variant or mutagenized form maintains binding to theaminophospholipid or anionic phospholipid, preferably PS.

[0349] In one particular embodiment, the invention provides an antibody,or antigen binding region thereof, in which the framework regions of the3G4 antibody (ATCC 4545) have been changed from mouse to a human IgG,such as human IgG₁ or other IgG subclass to reduce immunogenicity inhumans. In other embodiments, the sequences of the 3G4 antibody (ATCC4545) are examined for the presence of T-cell epitopes, as is known inthe art. The underlying sequence can then be changed to remove T-cellepitopes, i.e., to “deimmunize” the antibody.

[0350] The availability of the DNA and amino acid sequences of the Vhand Vκ chains of the 3G4 antibody (SEQ ID NO: 1, 2, 3 and 4) means thata range of antibodies can now be prepared using CDR technologies. Inparticular, random mutations are made in the CDRs and the productsscreened to identify antibodies with higher affinities and/or higherspecificities. Such mutagenesis and selection is routinely practiced inthe antibody arts. It is particularly suitable for use in the presentinvention, given the advantageous screening techniques disclosed herein.

[0351] These techniques are used to generate antibody variants withimproved biological properties relative to the parent antibody fromwhich they are prepared, such as the 9D2 and 3G4 (ATCC 4545) antibodies.Such variants, or second generation compounds, are typicallysubstitutional variants involving one or more substituted hypervariableregion residues of a parent antibody. A convenient way for generatingsuch substitutional variants is affinity maturation using phage display.

[0352] In affinity maturation using phage display, several hypervariableregion sites (e.g. 6-7 sites) are mutated to generate all possible aminosubstitutions at each site. The antibody variants thus generated aredisplayed in a monovalent fashion from filamentous phage particles asfusions to the gene III product of M13 packaged within each particle.The phage-displayed variants are then screened for their biologicalactivity (e.g. binding affinity) as herein disclosed. In order toidentify candidate hypervariable region sites for modification, alaninescanning mutagenesis can be performed to identify hypervariable regionresidues contributing significantly to antigen binding.

[0353] CDR shuffling and implantation technologies can also be used withthe antibodies of the present invention, preferably the 9D2 and 3G4(ATCC 4545) antibodies. CDR shuffling inserts CDR sequences into aspecific framework region (Jirholt et al., 1998, specificallyincorporated herein by reference). CDR implantation techniques permitthe random combination of CDR sequences into a single master framework(Soderlind et al., 1999, 2000, each specifically incorporated herein byreference). Using such techniques, the CDR sequences of the 3G4 (ATCC4545) antibody, for example, are mutagenized to create a plurality ofdifferent sequences, which are incorporated into a scaffold sequence andthe resultant antibody variants screened for desired characteristics,e.g., higher affinity.

[0354] In light of the information in the present disclosure, theantigen binding fragment of the antibodies, preferably the 9D2 and 3G4(ATCC 4545) antibodies, can also be minimized, giving enhancedstability. This can be achieved by preparing single domain bindingproteins based upon immunoglobulin VH and VH-like domains (Nuttall etal., 2000, specifically incorporated herein by reference).

[0355] Alternatively, or in addition, the crystal structure of theantigen-antibody complex can be delineated and analyzed to identifycontact points between the antibody and target aminophospholipid oranionic phospholipid, e.g., PS. Such contact residues and neighboringresidues are candidates for substitution. Once such variants aregenerated, the panel of variants is subjected to screening, as describedherein, and antibodies with analogous but different or even superiorproperties in one or more relevant assays are selected for furtherdevelopment.

[0356] D3. Camelized Antibodies

[0357] Further examples of antibodies of the invention are “camelized”antibodies. Antibodies from camels and llamas (Camelidae, camelids)include a unique kind of antibody, which is devoid of light chains andthus formed by heavy chains only. These have been termed “camelizedantibodies”. The antigen-binding site of such antibodies is one singledomain, referred to as V_(HH) (VHH).

[0358] As the DNA and amino acid sequences of the Vh and Vκ0 chains ofthe 3G4 (ATCC 4545) antibody are provided herein (SEQ ID NOs:1, 2, 3 and4), camelized versions of the 3G4 antibody can also be prepared.Mutations and structural adaptations can be made to reshape a V_(H) of aV_(H)-V_(L) pair into a single-domain VHH with retention of a sufficientvariability (Muyldermans et al., 2001, specifically incorporated hereinby reference). Such V_(HH) constructs are small, robust and efficientrecognition units (Riechmann and Muyldermans, 1999) with potentantigen-binding capacity, which can provide the further advantage ofinteracting with novel epitopes that are inaccessible to conventionalV_(H)-V_(L) pairs. Thus, camelised antibodies are akin to Fv fragments,but can have additional benefits.

[0359] U.S. Pat. No. 5,800,988, U.S. Pat. No. 6,005,079, PCT applicationNo. WO 94/04678, PCT application No. WO 94/25591, Riechmann andMuyldermans (1999) and Muyldermans et al. (2001) are each specificallyincorporated herein by reference for the purpose of even furtherdescribing and enabling the production of camelized antibodies.Accordingly, the CDR from the 3G4 antibody can be grafted on theframework of the variable domain of the heavy chain immunoglobulin ofthe Camelidae antibody.

[0360] D4. CDR Sequences

[0361] Further aspects of the invention therefore concern isolated DNAsegments and recombinant vectors encoding CDR regions of antibody heavyand light chains, such as 9D2 and 3G4, and preferably 3G4 (ATCC 4545),heavy and light chains, and the creation and use of recombinant hostcells and phage through the application of DNA technology, which expresssuch CDR regions.

[0362] The invention thus provides an isolated polynucleotide thatcontains a nucleotide sequence that encodes at least one CDR of theantibody produced by the hybridoma deposited as ATCC 4545. The inventionfurther provides an isolated polynucleotide that contains a nucleotidesequence that encodes a CDR, antibody, or antigen binding regionthereof, which binds to at least a first aminophospholipid or anionicphospholipid, preferably PS, and which comprises at least one CDR of theantibody produced by the hybridoma deposited as ATCC 4545.

[0363] Further aspects of the invention concern an isolatedpolynucleotide that contains a nucleotide sequence that encodes at leastone CDR that has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4,or a variant or mutagenized form thereof. Other aspects of the inventionconcern an isolated polynucleotide that contains a nucleotide sequencethat encodes a CDR, antibody, or antigen binding region thereof, whichbinds to at least a first aminophospholipid or anionic phospholipid,preferably PS, and which comprises at least one CDR with the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:4, or a variant or mutagenized formthereof, wherein such a variant or mutagenized form maintains binding tothe aminophospholipid or anionic phospholipid, preferably PS.

[0364] In other aspects of the invention, the isolated polynucleotidecontains the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, or avariant or mutagenized form thereof. In particular, the isolatedpolynucleotide contains the nucleotide sequence of SEQ ID NO:1 or SEQ IDNO:3, or a variant or mutagenized form thereof, which nucleotidesequence encodes a CDR, antibody, or antigen binding region thereof thatbinds to at least a first aminophospholipid or anionic phospholipid,preferably PS, wherein any such variant or mutagenized form maintainsbinding to the aminophospholipid or anionic phospholipid, preferably PS.

[0365] The present invention thus concerns polynucleotide and DNAsegments, isolatable from any mammal, preferably, human or murine, thatare free from total genomic DNA and are capable of expressing CDRregions of anti-anionic phospholipid or anti-aminophospholipid antibodyheavy and light chains, such as 9D2 and 3G4, and preferably 3G4 (ATCC4545), heavy and light chains. As used herein, the terms “polynucleotidesegment” and “DNA segment” refer to polynucleotides and DNA moleculesthat have been isolated free of total genomic DNA of a particularspecies. Included within the term “polynucleotide segment” and “DNAsegment”, are DNA segments and smaller fragments of such segments, andalso recombinant vectors, including, for example, plasmids, cosmids,phage, viruses, and the like.

[0366] Similarly, a DNA segment comprising a coding segment or isolatedgene portion encoding purified CDR regions of anti-anionic phospholipidor anti-aminophospholipid antibody heavy and light chains, such as 9D2and 3G4, and preferably 3G4, heavy and light chains, refers to a DNAsegment including such coding sequences and, in certain aspects,regulatory sequences, isolated substantially away from other naturallyoccurring genes or protein encoding sequences. In this respect, the term“gene” is used for simplicity to refer to a functional protein,polypeptide or peptide encoding unit. As will be understood by those inthe art, this functional term includes the native antibody-encodingsequences and smaller engineered segments that express, or may beadapted to express, suitable antigen binding proteins, polypeptides orpeptides.

[0367] “Isolated substantially away from other coding sequences” meansthat the coding segment or isolated gene portion of interest forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or cDNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to the segment by the hand of man.

[0368] In particular embodiments, the invention concerns isolated codingsegments or isolated gene portions and recombinant vectors incorporatingDNA sequences that encode CDR regions of anti-anionic phospholipid oranti-aminophospholipid antibody heavy and light chains, such as 9D2 and3G4, and preferably 3G4, heavy and light chains, that comprise at leasta first sequence region that includes an amino acid sequence region ofat least about 75%, more preferably, at least about 80%, morepreferably, at least about 85%, more preferably, at least about 90%,91%, 92%, 93%, 94%, and most preferably, at least about 95%, 96%, 97%,98% or 99% or so amino acid sequence identity to the amino acid sequenceof SEQ ID NO:2 or SEQ ID NO:4; wherein said CDR regions at leastsubstantially maintain the biological properties of the CDR regions ofamino acid sequences SEQ ID NO:2 or SEQ ID NO:2.

[0369] As disclosed herein, the sequences may comprise certainbiologically functional equivalent amino acids or “conservativesubstitutions”. Other sequences may comprise functionally non-equivalentamino acids or “non-conservative substitutions” deliberately engineeredto improve the properties of the CDR or antibody containing the CDR, asis known those of ordinary skill in the art and further describedherein.

[0370] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, and yet still correspondto a sequence of the invention, so long as the sequence meets thecriteria set forth above, preferably including the maintenance orimprovement of biological protein activity where protein expression isconcerned. The addition of terminal sequences includes variousnon-coding sequences flanking either of the 5′ or 3′ portions of thecoding region, and also control regions.

[0371] The nucleic acid segments of the present invention may thus becombined with other DNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol.

[0372] Recombinant vectors therefore form further aspects of the presentinvention. Particularly useful vectors are contemplated to be thosevectors in which the coding portion of the DNA segment is positionedunder the control of a promoter. Generally, although not exclusively, arecombinant or heterologous promoter will be employed, i.e., a promoternot normally associated with coding sequences in their naturalenvironment. Such promoters may include bacterial, viral, eukaryotic andmammalian promoters, so long as the promoter effectively directs theexpression of the DNA segment in the cell type, organism, or evenanimal, chosen for expression.

[0373] The use of promoter and cell type combinations for proteinexpression is known to those of skill in the art of molecular biology.The promoters employed may be constitutive, or inducible, and can beused under the appropriate conditions to direct high level expression ofthe introduced DNA segment, such as is advantageous in the large-scaleproduction of recombinant proteins or peptides.

[0374] The expression of the nucleic acid sequences of the invention maybe conveniently achieved by any one or more standard techniques knownthose of ordinary skill in the art and further described herein. Forexample, the later description of the recombinant expression of fusionproteins applies equally well to antibodies and antibody fragments thatare not operatively associated with another coding sequence at thenucleic acid level.

[0375] E. Further Antibody Preparation Techniques

[0376] E1. Antibodies from Phagemid Libraries

[0377] Recombinant technology now allows the preparation of antibodieshaving the desired specificity from recombinant genes encoding a rangeof antibodies (Van Dijk et al., 1989; incorporated herein by reference).Certain recombinant techniques involve the isolation of the antibodygenes by immunological screening of combinatorial immunoglobulin phageexpression libraries prepared from RNA isolated from the spleen of animmunized animal (Morrison et al., 1986; Winter and Milstein, 1991;Barbas et al., 1992; each incorporated herein by reference).

[0378] For such methods, combinatorial immunoglobulin phagemid librariesare prepared from RNA isolated from the spleen of the immunized animal,and phagemids expressing appropriate antibodies are selected by panningusing cells expressing the antigen and control cells. The advantages ofthis approach over conventional hybridoma techniques are thatapproximately 10⁴ times as many antibodies can be produced and screenedin a single round, and that new specificities are generated by H and Lchain combination, which further increases the percentage of appropriateantibodies generated.

[0379] One method for the generation of a large repertoire of diverseantibody molecules in bacteria utilizes the bacteriophage lambda as thevector (Huse et al., 1989; incorporated herein by reference). Productionof antibodies using the lambda vector involves the cloning of heavy andlight chain populations of DNA sequences into separate starting vectors.The vectors are subsequently combined randomly to form a single vectorthat directs the co-expression of heavy and light chains to formantibody fragments. The heavy and light chain DNA sequences are obtainedby amplification, preferably by PCR™ or a related amplificationtechnique, of mRNA isolated from spleen cells (or hybridomas thereof)from an animal that has been immunized with a selected antigen. Theheavy and light chain sequences are typically amplified using primersthat incorporate restriction sites into the ends of the amplified DNAsegment to facilitate cloning of the heavy and light chain segments intothe starting vectors.

[0380] Another method for the generation and screening of largelibraries of wholly or partially synthetic antibody combining sites, orparatopes, utilizes display vectors derived from filamentous phage suchas M13, fl or fd. These filamentous phage display vectors, referred toas “phagemids”, yield large libraries of monoclonal antibodies havingdiverse and novel immunospecificities. The technology uses a filamentousphage coat protein membrane anchor domain as a means for linkinggene-product and gene during the assembly stage of filamentous phagereplication, and has been used for the cloning and expression ofantibodies from combinatorial libraries (Kang et al., 1991; Barbas etal., 1991; each incorporated herein by reference).

[0381] This general technique for filamentous phage display is describedin U.S. Pat. No. 5,658,727, incorporated herein by reference. In a mostgeneral sense, the method provides a system for the simultaneous cloningand screening of pre-selected ligand-binding specificities from antibodygene repertoires using a single vector system. Screening of isolatedmembers of the library for a pre-selected ligand-binding capacity allowsthe correlation of the binding capacity of an expressed antibodymolecule with a convenient means to isolate the gene that encodes themember from the library.

[0382] Linkage of expression and screening is accomplished by thecombination of targeting of a fusion polypeptide into the periplasm of abacterial cell to allow assembly of a functional antibody, and thetargeting of a fusion polypeptide onto the coat of a filamentous phageparticle during phage assembly to allow for convenient screening of thelibrary member of interest. Periplasmic targeting is provided by thepresence of a secretion signal domain in a fusion polypeptide. Targetingto a phage particle is provided by the presence of a filamentous phagecoat protein membrane anchor domain (i.e., a cpIII- or cpVIII-derivedmembrane anchor domain) in a fusion polypeptide.

[0383] The diversity of a filamentous phage-based combinatorial antibodylibrary can be increased by shuffling of the heavy and light chaingenes, by altering one or more of the complementarity determiningregions of the cloned heavy chain genes of the library, or byintroducing random mutations into the library by error-prone polymerasechain reactions. Additional methods for screening phagemid libraries aredescribed in U.S. Pat. Nos. 5,580,717; 5,427,908; 5,403,484; and5,223,409, each incorporated herein by reference.

[0384] Another method for the screening of large combinatorial antibodylibraries has been developed, utilizing expression of populations ofdiverse heavy and light chain sequences on the surface of a filamentousbacteriophage, such as M13, fl or fd (U.S. Pat. No. 5,698,426;incorporated herein by reference). Two populations of diverse heavy (Hc)and light (Lc) chain sequences are synthesized by polymerase chainreaction (PCR™). These populations are cloned into separate M13-basedvector containing elements necessary for expression. The heavy chainvector contains a gene VIII (gVIII) coat protein sequence so thattranslation of the heavy chain sequences produces gVIII-Hc fusionproteins. The populations of two vectors are randomly combined such thatonly the vector portions containing the Hc and Lc sequences are joinedinto a single circular vector.

[0385] The combined vector directs the co-expression of both Hc and Lcsequences for assembly of the two polypeptides and surface expression onM13 (U.S. Pat. No. 5,698,426; incorporated herein by reference). Thecombining step randomly brings together different Hc and Lc encodingsequences within two diverse populations into a single vector. Thevector sequences donated from each independent vector are necessary forproduction of viable phage. Also, since the pseudo gVIII sequences arecontained in only one of the two starting vectors, co-expression offunctional antibody fragments as Lc associated gVIII-Hc fusion proteinscannot be accomplished on the phage surface until the vector sequencesare linked in the single vector.

[0386] Surface expression of the antibody library is performed in anamber suppressor strain. An amber stop codon between the Hc sequence andthe gVIII sequence unlinks the two components in a non-suppressorstrain. Isolating the phage produced from the non-suppressor strain andinfecting a suppressor strain will link the Hc sequences to the gVIIIsequence during expression. Culturing the suppressor strain afterinfection allows the coexpression on the surface of M13 of all antibodyspecies within the library as gVIII fusion proteins (gVIII-Fab fusionproteins). Alternatively, the DNA can be isolated from thenon-suppressor strain and then introduced into a suppressor strain toaccomplish the same effect.

[0387] The surface expression library is screened for specific Fabfragments that bind preselected molecules by standard affinity isolationprocedures. Such methods include, for example, panning (Parmley andSmith, 1988; incorporated herein by reference), affinity chromatographyand solid phase blotting procedures. Panning is preferred, because hightiters of phage can be screened easily, quickly and in small volumes.Furthermore, this procedure can select minor Fab fragments specieswithin the population, which otherwise would have been undetectable, andamplified to substantially homogenous populations. The selected Fabfragments can be characterized by sequencing the nucleic acids encodingthe polypeptides after amplification of the phage population.

[0388] Another method for producing diverse libraries of antibodies andscreening for desirable binding specificities is described in U.S. Pat.No. 5,667,988 and U.S. Pat. No. 5,759,817, each incorporated herein byreference. The method involves the preparation of libraries ofheterodimeric immunoglobulin molecules in the form of phagemid librariesusing degenerate oligonucleotides and primer extension reactions toincorporate the degeneracies into the CDR regions of the immunoglobulinvariable heavy and light chain variable domains, and display of themutagenized polypeptides on the surface of the phagemid. Thereafter, thedisplay protein is screened for the ability to bind to a preselectedantigen.

[0389] The method for producing a heterodimeric immunoglobulin moleculegenerally involves (1) introducing a heavy or light chain Vregion-coding gene of interest into the phagemid display vector; (2)introducing a randomized binding site into the phagemid display proteinvector by primer extension with an oligonucleotide containing regions ofhomology to a CDR of the antibody V region gene and containing regionsof degeneracy for producing randomized coding sequences to form a largepopulation of display vectors each capable of expressing differentputative binding sites displayed on a phagemid surface display protein;(3) expressing the display protein and binding site on the surface of afilamentous phage particle; and (4) isolating (screening) thesurface-expressed phage particle using affinity techniques such aspanning of phage particles against a preselected antigen, therebyisolating one or more species of phagemid containing a display proteincontaining a binding site that binds a preselected antigen.

[0390] A further variation of this method for producing diverselibraries of antibodies and screening for desirable bindingspecificities is described in U.S. Pat. No. 5,702,892, incorporatedherein by reference. In this method, only heavy chain sequences areemployed, the heavy chain sequences are randomized at all nucleotidepositions which encode either the CDRI or CDRIII hypervariable region,and the genetic variability in the CDRs is generated independent of anybiological process.

[0391] In the method, two libraries are engineered to geneticallyshuffle oligonucleotide motifs within the framework of the heavy chaingene structure. Through random mutation of either CDRI or CDRIII, thehypervariable regions of the heavy chain gene were reconstructed toresult in a collection of highly diverse sequences. The heavy chainproteins encoded by the collection of mutated gene sequences possessedthe potential to have all of the binding characteristics of animmunoglobulin while requiring only one of the two immunoglobulinchains.

[0392] Specifically, the method is practiced in the absence of theimmunoglobulin light chain protein. A library of phage displayingmodified heavy chain proteins is incubated with an immobilized ligand toselect clones encoding recombinant proteins that specifically bind theimmobilized ligand. The bound phage are then dissociated from theimmobilized ligand and amplified by growth in bacterial host cells.Individual viral plaques, each expressing a different recombinantprotein, are expanded, and individual clones can then be assayed forbinding activity.

[0393] E2. Antibodies from Human Lymphocytes

[0394] Antibodies against phospholipids occur in the human population.However, these antibodies are typically associated with disease andtheir use in the present invention should preferably be avoided.However, human lymphocytes from healthy subjects can be used asappropriate as starting materials for generating an antibody for use inthe invention.

[0395] In vitro immunization, or antigen stimulation, may also be usedto generate a human antibody for use in the present invention. Suchtechniques can be used to stimulate peripheral blood lymphocytes fromnormal, healthy subjects simply by stimulating antibody-producing cellswith anionic phospholipids and aminophospholipids in vitro.

[0396] Such “in vitro immunization” involves antigen-specific activationof non-immunized B lymphocytes, generally within a mixed population oflymphocytes (mixed lymphocyte cultures, MLC). In vitro immunizations mayalso be supported by B cell growth and differentiation factors andlymphokines. The antibodies produced by these methods are often IgMantibodies (Borrebaeck and Moller, 1986; incorporated herein byreference).

[0397] Another method has been described (U.S. Pat. No. 5,681,729,incorporated herein by reference) wherein human lymphocytes that mainlyproduce IgG (or IgA) antibodies can be obtained. The method involves, ina general sense, transplanting human lymphocytes to an immunodeficientanimal so that the human lymphocytes “take” in the animal body;immunizing the animal with a desired antigen, so as to generate humanlymphocytes producing an antibody specific to the antigen; andrecovering the human lymphocytes producing the antibody from the animal.The human lymphocytes thus produced can be used to produce a monoclonalantibody by immortalizing the human lymphocytes producing the antibody,cloning the obtained immortalized human-originated lymphocytes producingthe antibody, and recovering a monoclonal antibody specific to thedesired antigen from the cloned immortalized human-originatedlymphocytes.

[0398] The immunodeficient animals that may be employed in thistechnique are those that do not exhibit rejection when human lymphocytesare transplanted to the animals. Such animals may be artificiallyprepared by physical, chemical or biological treatments. Anyimmunodeficient animal may be employed. The human lymphocytes may beobtained from human peripheral blood, spleen, lymph nodes, tonsils orthe like.

[0399] The “taking” of the transplanted human lymphocytes in the animalscan be attained by merely administering the human lymphocytes to theanimals. The administration route is not restricted and may be, forexample, subcutaneous, intravenous or intraperitoneal. The dose of thehuman lymphocytes is not restricted, and can usually be 10⁶ to 10⁸lymphocytes per animal. The immunodeficient animal is then immunizedwith the desired antigen.

[0400] After the immunization, human lymphocytes are recovered from theblood, spleen, lymph nodes or other lymphatic tissues by anyconventional method. For example, mononuclear cells can be separated bythe Ficoll-Hypaque (specific gravity: 1.077) centrifugation method, andthe monocytes removed by the plastic dish adsorption method. Thecontaminating cells originating from the immunodeficient animal may beremoved by using an antiserum specific to the animal cells. Theantiserum may be obtained by, for example, immunizing a second, distinctanimal with the spleen cells of the immunodeficient animal, andrecovering serum from the distinct immunized animal. The treatment withthe antiserum may be carried out at any stage. The human lymphocytes mayalso be recovered by an immunological method employing a humanimmunoglobulin expressed on the cell surface as a marker.

[0401] By these methods, human lymphocytes mainly producing IgG and IgAantibodies specific to one or more selected anionic phospholipids andaminophospholipids can be obtained. Monoclonal antibodies are thenobtained from the human lymphocytes by immortalization, selection, cellgrowth and antibody production.

[0402] E3. Transgenic Mice Containing Human Antibody Libraries

[0403] Recombinant technology is now available for the preparation ofantibodies. In addition to the combinatorial immunoglobulin phageexpression libraries disclosed above, another molecular cloning approachis to prepare antibodies from transgenic mice containing human antibodylibraries. Such techniques are described in U.S. Pat. No. 5,545,807,incorporated herein by reference.

[0404] In a most general sense, these methods involve the production ofa transgenic animal that has inserted into its germline genetic materialthat encodes for at least part of an immunoglobulin of human origin orthat can rearrange to encode a repertoire of immunoglobulins. Theinserted genetic material may be produced from a human source, or may beproduced synthetically. The material may code for at least part of aknown immunoglobulin or may be modified to code for at least part of analtered immunoglobulin.

[0405] The inserted genetic material is expressed in the transgenicanimal, resulting in production of an immunoglobulin derived at least inpart from the inserted human immunoglobulin genetic material. It isfound the genetic material is rearranged in the transgenic animal, sothat a repertoire of immunoglobulins with part or parts derived frominserted genetic material may be produced, even if the inserted geneticmaterial is incorporated in the germline in the wrong position or withthe wrong geometry.

[0406] The inserted genetic material may be in the form of DNA clonedinto prokaryotic vectors such as plasmids and/or cosmids. Larger DNAfragments are inserted using yeast artificial chromosome vectors (Burkeet al., 1987; incorporated herein by reference), or by introduction ofchromosome fragments (Richer and Lo, 1989; incorporated herein byreference). The inserted genetic material may be introduced to the hostin conventional manner, for example by injection or other proceduresinto fertilized eggs or embryonic stem cells.

[0407] In preferred aspects, a host animal that initially does not carrygenetic material encoding immunoglobulin constant regions is utilized,so that the resulting transgenic animal will use only the inserted humangenetic material when producing immunoglobulins. This can be achievedeither by using a naturally occurring mutant host lacking the relevantgenetic material, or by artificially making mutants e.g., in cell linesultimately to create a host from which the relevant genetic material hasbeen removed.

[0408] Where the host animal carries genetic material encodingimmunoglobulin constant regions, the transgenic animal will carry thenaturally occurring genetic material and the inserted genetic materialand will produce immunoglobulins derived from the naturally occurringgenetic material, the inserted genetic material, and mixtures of bothtypes of genetic material. In this case the desired immunoglobulin canbe obtained by screening hybridomas derived from the transgenic animal,e.g., by exploiting the phenomenon of allelic exclusion of antibody geneexpression or differential chromosome loss.

[0409] Once a suitable transgenic animal has been prepared, the animalis simply immunized with the desired immunogen. Depending on the natureof the inserted material, the animal may produce a chimericimmunoglobulin, e.g. of mixed mouse/human origin, where the geneticmaterial of foreign origin encodes only part of the immunoglobulin; orthe animal may produce an entirely foreign immunoglobulin, e.g. ofwholly human origin, where the genetic material of foreign originencodes an entire immunoglobulin.

[0410] Polyclonal antisera may be produced from the transgenic animalfollowing immunization. Immunoglobulin-producing cells may be removedfrom the animal to produce the immunoglobulin of interest. Preferably,monoclonal antibodies are produced from the transgenic animal, e.g., byfusing spleen cells from the animal with myeloma cells and screening theresulting hybridomas to select those producing the desired antibody.Suitable techniques for such processes are described herein.

[0411] In an alternative approach, the genetic material may beincorporated in the animal in such a way that the desired antibody isproduced in body fluids such as serum or external secretions of theanimal, such as milk, colostrum or saliva. For example, by inserting invitro genetic material encoding for at least part of a humanimmunoglobulin into a gene of a mammal coding for a milk protein andthen introducing the gene to a fertilized egg of the mammal, e.g., byinjection, the egg may develop into an adult female mammal producingmilk containing immunoglobulin derived at least in part from theinserted human immunoglobulin genetic material. The desired antibody canthen be harvested from the milk. Suitable techniques for carrying outsuch processes are known to those skilled in the art.

[0412] The foregoing transgenic animals are usually employed to producehuman antibodies of a single isotype, more specifically an isotype thatis essential for B cell maturation, such as IgM and possibly IgD.Another preferred method for producing human antibodies is described inU.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016;and 5,770,429; each incorporated by reference, wherein transgenicanimals are described that are capable of switching from an isotypeneeded for B cell development to other isotypes.

[0413] In the development of a B lymphocyte, the cell initially producesIgM with a binding specificity determined by the productively rearrangedV_(H) and V_(L) regions. Subsequently, each B cell and its progeny cellssynthesize antibodies with the same L and H chain V regions, but theymay switch the isotype of the H chain. The use of mu or delta constantregions is largely determined by alternate splicing, permitting IgM andIgD to be coexpressed in a single cell. The other heavy chain isotypes(gamma, alpha, and epsilon) are only expressed natively after a generearrangement event deletes the C mu and C delta exons. This generearrangement process, termed isotype switching, typically occurs byrecombination between so called switch segments located immediatelyupstream of each heavy chain gene (except delta). The individual switchsegments are between 2 and 10 kb in length, and consist primarily ofshort repeated sequences.

[0414] For these reasons, it is preferable that transgenes incorporatetranscriptional regulatory sequences within about 1-2 kb upstream ofeach switch region that is to be utilized for isotype switching. Thesetranscriptional regulatory sequences preferably include a promoter andan enhancer element, and more preferably include the 5′ flanking (i.e.,upstream) region that is naturally associated (i.e., occurs in germlineconfiguration) with a switch region. Although a 5′ flanking sequencefrom one switch region can be operably linked to a different switchregion for transgene construction, in some embodiments it is preferredthat each switch region incorporated in the transgene construct have the5′ flanking region that occurs immediately upstream in the naturallyoccurring germline configuration. Sequence information relating toimmunoglobulin switch region sequences is known (Mills et al., 1990;Sideras et al., 1989; each incorporated herein by reference).

[0415] In the method described in U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,661,016; and 5,770,429, the human immunoglobulintransgenes contained within the transgenic animal function correctlythroughout the pathway of B-cell development, leading to isotypeswitching. Accordingly, in this method, these transgenes are constructedso as to produce isotype switching and one or more of the following: (1)high level and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

[0416] An important requirement for transgene function is the generationof a primary antibody repertoire that is diverse enough to trigger asecondary immune response for a wide range of antigens. The rearrangedheavy chain gene consists of a signal peptide exon, a variable regionexon and a tandem array of multi-domain constant region regions, each ofwhich is encoded by several exons. Each of the constant region genesencode the constant portion of a different class of immunoglobulins.During B-cell development, V region proximal constant regions aredeleted leading to the expression of new heavy chain classes. For eachheavy chain class, alternative patterns of RNA splicing give rise toboth transmembrane and secreted immunoglobulins.

[0417] The human heavy chain locus consists of approximately 200 V genesegments spanning 2 Mb, approximately 30 D gene segments spanning about40 kb, six J segments clustered within a 3 kb span, and nine constantregion gene segments spread out over approximately 300 kb. The entirelocus spans approximately 2.5 Mb of the distal portion of the long armof chromosome 14. Heavy chain transgene fragments containing members ofall six of the known VH families, the D and J gene segments, as well asthe mu, delta, gamma 3, gamma 1 and alpha 1 constant regions are known(Berman et al., 1988; incorporated herein by reference). Genomicfragments containing all of the necessary gene segments and regulatorysequences from a human light chain locus is similarly constructed.

[0418] The expression of successfully rearranged immunoglobulin heavyand light transgenes usually has a dominant effect by suppressing therearrangement of the endogenous immunoglobulin genes in the transgenicnonhuman animal. However, in certain embodiments, it is desirable toeffect complete inactivation of the endogenous Ig loci so that hybridimmunoglobulin chains comprising a human variable region and a non-human(e.g., murine) constant region cannot be formed, for example bytrans-switching between the transgene and endogenous Ig sequences. Usingembryonic stem cell technology and homologous recombination, theendogenous immunoglobulin repertoire can be readily eliminated. Inaddition, suppression of endogenous Ig genes may be accomplished using avariety of techniques, such as antisense technology.

[0419] In other aspects of the invention, it may be desirable to producea trans-switched immunoglobulin. Antibodies comprising such chimerictrans-switched immunoglobulins can be used for a variety of applicationswhere it is desirable to have a non-human (e.g., murine) constantregion, e.g., for retention of effector functions in the host. Thepresence of a murine constant region can afford advantages over a humanconstant region, for example, to provide murine effector functions(e.g., ADCC, murine complement fixation) so that such a chimericantibody may be tested in a mouse disease model. Subsequent to theanimal testing, the human variable region encoding sequence may beisolated, e.g., by PCR amplification or cDNA cloning from the source(hybridoma clone), and spliced to a sequence encoding a desired humanconstant region to encode a human sequence antibody more suitable forhuman therapeutic use.

[0420] E4. Humanized Antibodies

[0421] Human antibodies generally have at least three potentialadvantages for use in human therapy. First, because the effector portionis human, it may interact better with the other parts of the humanimmune system, e.g., to destroy target cells more efficiently bycomplement-dependent cytotoxicity (CDC) or antibody-dependent cellularcytotoxicity (ADCC). Second, the human immune system should notrecognize the antibody as foreign. Third, the half-life in the humancirculation will be similar to naturally occurring human antibodies,allowing smaller and less frequent doses to be given.

[0422] Various methods for preparing human antibodies are providedherein. In addition to human antibodies, “humanized” antibodies havemany advantages. “Humanized” antibodies are generally chimeric or mutantmonoclonal antibodies from mouse, rat, hamster, rabbit or other species,bearing human constant and/or variable region domains or specificchanges. Techniques for generating a so-called “humanized” antibody arewell known to those of skill in the art.

[0423] Humanized antibodies also share the foregoing advantages. First,the effector portion is still human. Second, the human immune systemshould not recognize the framework or constant region as foreign, andtherefore the antibody response against such an injected antibody shouldbe less than against a totally foreign mouse antibody. Third, injectedhumanized antibodies, as opposed to injected mouse antibodies, willpresumably have a half-life more similar to naturally occurring humanantibodies, also allowing smaller and less frequent doses.

[0424] A number of methods have been described to produce humanizedantibodies. Controlled rearrangement of antibody domains joined throughprotein disulfide bonds to form new, artificial protein molecules or“chimeric” antibodies can be utilized (Konieczny et al., 1981;incorporated herein by reference). Recombinant DNA technology can alsobe used to construct gene fusions between DNA sequences encoding mouseantibody variable light and heavy chain domains and human antibody lightand heavy chain constant domains (Morrison et al., 1984; incorporatedherein by reference).

[0425] DNA sequences encoding the antigen binding portions orcomplementarity determining regions (CDR's) of murine monoclonalantibodies can be grafted by molecular means into the DNA sequencesencoding the frameworks of human antibody heavy and light chains (Joneset al., 1986; Riechmann et al., 1988; each incorporated herein byreference). The expressed recombinant products are called “reshaped” orhumanized antibodies, and comprise the framework of a human antibodylight or heavy chain and the antigen recognition portions, CDR's, of amurine monoclonal antibody.

[0426] Another method for producing humanized antibodies is described inU.S. Pat. No. 5,639,641, incorporated herein by reference. The methodprovides, via resurfacing, humanized rodent antibodies that haveimproved therapeutic efficacy due to the presentation of a human surfacein the variable region. In the method: (1) position alignments of a poolof antibody heavy and light chain variable regions is generated to givea set of heavy and light chain variable region framework surface exposedpositions, wherein the alignment positions for all variable regions areat least about 98% identical; (2) a set of heavy and light chainvariable region framework surface exposed amino acid residues is definedfor a rodent antibody (or fragment thereof); (3) a set of heavy andlight chain variable region framework surface exposed amino acidresidues that is most closely identical to the set of rodent surfaceexposed amino acid residues is identified; (4) the set of heavy andlight chain variable region framework surface exposed amino acidresidues defined in step (2) is substituted with the set of heavy andlight chain variable region framework surface exposed amino acidresidues identified in step (3), except for those amino acid residuesthat are within 5 Å of any atom of any residue of the complementaritydetermining regions of the rodent antibody; and (5) the humanized rodentantibody having binding specificity is produced.

[0427] A similar method for the production of humanized antibodies isdescribed in U.S. Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and5,530,101, each incorporated herein by reference. These methods involveproducing humanized immunoglobulins having one or more complementaritydetermining regions (CDR's) and possible additional amino acids from adonor immunoglobulin and a framework region from an accepting humanimmunoglobulin. Each humanized immunoglobulin chain usually comprises,in addition to the CDR's, amino acids from the donor immunoglobulinframework that are capable of interacting with the CDR's to effectbinding affinity, such as one or more amino acids that are immediatelyadjacent to a CDR in the donor immunoglobulin or those within about 3 Åas predicted by molecular modeling. The heavy and light chains may eachbe designed by using any one, any combination, or all of the variousposition criteria described in U.S. Pat. Nos. 5,693,762; 5,693,761;5,585,089; and 5,530,101, each incorporated herein by reference. Whencombined into an intact antibody, the humanized immunoglobulins aresubstantially non-immunogenic in humans and retain substantially thesame affinity as the donor immunoglobulin to the original antigen.

[0428] An additional method for producing humanized antibodies isdescribed in U.S. Pat. Nos. 5,565,332 and 5,733,743, each incorporatedherein by reference. This method combines the concept of humanizingantibodies with the phagemid libraries also described in detail herein.In a general sense, the method utilizes sequences from the antigenbinding site of an antibody or population of antibodies directed againstan antigen of interest. Thus for a single rodent antibody, sequencescomprising part of the antigen binding site of the antibody may becombined with diverse repertoires of sequences of human antibodies thatcan, in combination, create a complete antigen binding site.

[0429] The antigen binding sites created by this process differ fromthose created by CDR grafting, in that only the portion of sequence ofthe original rodent antibody is likely to make contacts with antigen ina similar manner. The selected human sequences are likely to differ insequence and make alternative contacts with the antigen from those ofthe original binding site. However, the constraints imposed by bindingof the portion of original sequence to antigen and the shapes of theantigen and its antigen binding sites, are likely to drive the newcontacts of the human sequences to the same region or epitope of theantigen. This process has therefore been termed “epitope imprintedselection” (EIS).

[0430] Starting with an animal antibody, one process results in theselection of antibodies that are partly human antibodies. Suchantibodies may be sufficiently similar in sequence to human antibodiesto be used directly in therapy or after alteration of a few keyresidues. Sequence differences between the rodent component of theselected antibody with human sequences could be minimized by replacingthose residues that differ with the residues of human sequences, forexample, by site directed mutagenesis of individual residues, or by CDRgrafting of entire loops. However, antibodies with entirely humansequences can also be created. EIS therefore offers a method for makingpartly human or entirely human antibodies that bind to the same epitopeas animal or partly human antibodies respectively. In EIS, repertoiresof antibody fragments can be displayed on the surface of filamentousphase and the genes encoding fragments with antigen binding activitiesselected by binding of the phage to antigen.

[0431] Additional methods for humanizing antibodies contemplated for usein the present invention are described in U.S. Pat. Nos. 5,750,078;5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864;4,935,496; and 4,816,567, each incorporated herein by reference.

[0432] E5. Mutagenesis by PCR™

[0433] Site-specific mutagenesis is a technique useful in thepreparation of individual antibodies through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, incorporating one or more of theforegoing considerations, whether humanizing or not, by introducing oneor more nucleotide sequence changes into the DNA.

[0434] Although many methods are suitable for use in mutagenesis, theuse of the polymerase chain reaction (PCR™) is generally now preferred.This technology offers a quick and efficient method for introducingdesired mutations into a given DNA sequence. The following textparticularly describes the use of PCR™ to introduce point mutations intoa sequence, as may be used to change the amino acid encoded by the givensequence. Adaptations of this method are also suitable for introducingrestriction enzyme sites into a DNA molecule.

[0435] In this method, synthetic oligonucleotides are designed toincorporate a point mutation at one end of an amplified segment.Following PCR™, the amplified fragments are blunt-ended by treating withKlenow fragments, and the blunt-ended fragments are then ligated andsubcloned into a vector to facilitate sequence analysis.

[0436] To prepare the template DNA that one desires to mutagenize, theDNA is subcloned into a high copy number vector, such as pUC19, usingrestriction sites flanking the area to be mutated. Template DNA is thenprepared using a plasmid miniprep. Appropriate oligonucleotide primersthat are based upon the parent sequence, but which contain the desiredpoint mutation and which are flanked at the 5′ end by a restrictionenzyme site, are synthesized using an automated synthesizer. It isgenerally required that the primer be homologous to the template DNA forabout 15 bases or so. Primers may be purified by denaturingpolyacrylamide gel electrophoresis, although this is not absolutelynecessary for use in PCR™. The 5′ end of the oligonucleotides shouldthen be phosphorylated.

[0437] The template DNA should be amplified by PCR™, using theoligonucleotide primers that contain the desired point mutations. Theconcentration of MgCl₂ in the amplification buffer will generally beabout 15 mM. Generally about 20-25 cycles of PCR™ should be carried outas follows: denaturation, 35 sec. at 95° C.; hybridization, 2 min. at50° C.; and extension, 2 min. at 72° C. The PCR™ will generally includea last cycle extension of about 10 min. at 72° C. After the finalextension step, about 5 units of Klenow fragments should be added to thereaction mixture and incubated for a further 15 min. at about 30° C. Theexonuclease activity of the Klenow fragments is required to make theends flush and suitable for blunt-end cloning.

[0438] The resultant reaction mixture should generally be analyzed bynondenaturing agarose or acrylamide gel electrophoresis to verify thatthe amplification has yielded the predicted product. One would thenprocess the reaction mixture by removing most of the mineral oils,extracting with chloroform to remove the remaining oil, extracting withbuffered phenol and then concentrating by precipitation with 100%ethanol. Next, one should digest about half of the amplified fragmentswith a restriction enzyme that cuts at the flanking sequences used inthe oligonucleotides. The digested fragments are purified on a lowgelling/melting agarose gel.

[0439] To subclone the fragments and to check the point mutation, onewould subclone the two amplified fragments into an appropriatelydigested vector by blunt-end ligation. This would be used to transformE. coli, from which plasmid DNA could subsequently be prepared using aminiprep. The amplified portion of the plasmid DNA would then beanalyzed by DNA sequencing to confirm that the correct point mutationwas generated. This is important as Taq DNA polymerase can introduceadditional mutations into DNA fragments.

[0440] The introduction of a point mutation can also be effected usingsequential PCR™ steps. In this procedure, the two fragments encompassingthe mutation are annealed with each other and extended by mutuallyprimed synthesis. This fragment is then amplified by a second PCR™ step,thereby avoiding the blunt-end ligation required in the above protocol.In this method, the preparation of the template DNA, the generation ofthe oligonucleotide primers and the first PCR™ amplification areperformed as described above. In this process, however, the chosenoligonucleotides should be homologous to the template DNA for a stretchof between about 15 and about 20 bases and must also overlap with eachother by about 10 bases or more.

[0441] In the second PCR™ amplification, one would use each amplifiedfragment and each flanking sequence primer and carry PCR™ for betweenabout 20 and about 25 cycles, using the conditions as described above.One would again subclone the fragments and check that the point mutationwas correct by using the steps outlined above.

[0442] In using either of the foregoing methods, it is generallypreferred to introduce the mutation by amplifying as small a fragment aspossible. Of course, parameters such as the melting temperature of theoligonucleotide, as will generally be influenced by the GC content andthe length of the oligo, should also be carefully considered. Theexecution of these methods, and their optimization if necessary, will beknown to those of skill in the art, and are further described in variouspublications, such as Current Protocols in Molecular Biology, 1995,incorporated herein by reference.

[0443] When performing site-specific mutagenesis, Table A can beemployed as a reference. TABLE A Amino Acids Codons Alanine Ala A GCAGCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamicacid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGGGGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys KAAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0444] E6. Antibody Fragments and Derivatives

[0445] Irrespective of the source of the original antibody against ananionic phospholipid or aminophospholipids, either the intact antibody,antibody multimers, or any one of a variety of functional,antigen-binding regions of the antibody may be used in the presentinvention. Exemplary functional regions include scFv, Fv, Fab′, Fab andF(ab′)₂ fragments of antibodies. Techniques for preparing suchconstructs are well known to those in the art and are furtherexemplified herein.

[0446] The choice of antibody construct may be influenced by variousfactors. For example, prolonged half-life can result from the activereadsorption of intact antibodies within the kidney, a property of theFc piece of immunoglobulin. IgG based antibodies, therefore, areexpected to exhibit slower blood clearance than their Fab′ counterparts.However, Fab′ fragment-based compositions will generally exhibit bettertissue penetrating capability.

[0447] Antibody fragments can be obtained by proteolysis of the wholeimmunoglobulin by the non-specific thiol protease, papain. Papaindigestion yields two identical antigen-binding fragments, termed “Fabfragments”, each with a single antigen-binding site, and a residual “Fcfragment”.

[0448] Papain should first be activated by reducing the sulphydryl groupin the active site with cysteine, 2-mercaptoethanol or dithiothreitol.Heavy metals in the stock enzyme should be removed by chelation withEDTA (2 mM) to ensure maximum enzyme activity. Enzyme and substrate arenormally mixed together in the ratio of 1:100 by weight. Afterincubation, the reaction can be stopped by irreversible alkylation ofthe thiol group with iodoacetamide or simply by dialysis. Thecompleteness of the digestion should be monitored by SDS-PAGE and thevarious fractions separated by protein A-Sepharose or ion exchangechromatography.

[0449] The usual procedure for preparation of F(ab′)₂ fragments from IgGof rabbit and human origin is limited proteolysis by the enzyme pepsin.The conditions, 100× antibody excess w/w in acetate buffer at pH 4.5,37° C., suggest that antibody is cleaved at the C-terminal side of theinter-heavy-chain disulfide bond. Rates of digestion of mouse IgG mayvary with subclass and it may be difficult to obtain high yields ofactive F(ab′)₂ fragments without some undigested or completely degradedIgG. In particular, IgG_(2b) is highly susceptible to completedegradation. The other subclasses require different incubationconditions to produce optimal results, all of which is known in the art.

[0450] Pepsin treatment of intact antibodies yields an F(ab′)₂ fragmentthat has two antigen-combining sites and is still capable ofcross-linking antigen. Digestion of rat IgG by pepsin requiresconditions including dialysis in 0.1 M acetate buffer, pH 4.5, and thenincubation for four hours with 1% w/w pepsin; IgG₁ and IgG_(2a)digestion is improved if first dialyzed against 0.1 M formate buffer, pH2.8, at 4° C., for 16 hours followed by acetate buffer. IgG_(2b) givesmore consistent results with incubation in staphylococcal V8 protease(3% w/w) in 0.1 M sodium phosphate buffer, pH 7.8, for four hours at 37°C.

[0451] An Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain CH1 domain including one ormore cysteine(s) from the antibody hinge region. F(ab′)₂ antibodyfragments were originally produced as pairs of Fab′ fragments that havehinge cysteines between them. Other chemical couplings of antibodyfragments are also known.

[0452] An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen-recognition and binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,con-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

[0453] “Single-chain Fv” or “scFv” antibody fragments (now also known as“single chains”) comprise the V_(H) and V_(L) domains of an antibody,wherein these domains are present in a single polypeptide chain.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains that enables the sFv to form thedesired structure for antigen binding.

[0454] The following patents are specifically incorporated herein byreference for the purposes of even further supplementing the presentteachings regarding the preparation and use of functional,antigen-binding regions of antibodies, including scFv, Fv, Fab′, Fab andF(ab′)₂ fragments of antibodies: U.S. Pat. Nos. 5,855,866; 5,877,289;5,965,132; 6,093,399; 6,261,535 and 6,004,555. WO 98/45331 is alsoincorporated herein by reference for purposes including even furtherdescribing and teaching the preparation of variable, hypervariable andcomplementarity determining (CDR) regions of antibodies. Moreover, thesuccessful production of scFv constructs within the scope of the presentinvention is detailed in Example XIV.

[0455] “Diabodies” are small antibody fragments with two antigen-bindingsites, which fragments comprise a heavy chain variable domain (V_(H))connected to a light chain variable domain (V_(L)) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described in EP 404,097and WO 93/11161, each specifically incorporated herein by reference.“Linear antibodies”, which can be bispecific or monospecific, comprise apair of tandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) that form a pairof antigen binding regions, as described in Zapata et al. (1995),specifically incorporated herein by reference.

[0456] In using a Fab′ or antigen binding fragment of an antibody, withthe attendant benefits on tissue penetration, one may derive additionaladvantages from modifying the fragment to increase its half-life. Avariety of techniques may be employed, such as manipulation ormodification of the antibody molecule itself, and also conjugation toinert carriers. Any conjugation for the sole purpose of increasinghalf-life, rather than to deliver an agent to a target, should beapproached carefully in that Fab′ and other fragments are chosen topenetrate tissues. Nonetheless, conjugation to non-protein polymers,such PEG and the like, is contemplated.

[0457] Modifications other than conjugation are therefore based uponmodifying the structure of the antibody fragment to render it morestable, and/or to reduce the rate of catabolism in the body. Onemechanism for such modifications is the use of D-amino acids in place ofL-amino acids. Those of ordinary skill in the art will understand thatthe introduction of such modifications needs to be followed by rigoroustesting of the resultant molecule to ensure that it still retains thedesired biological properties. Further stabilizing modifications includethe use of the addition of stabilizing moieties to either the N-terminalor the C-terminal, or both, which is generally used to prolong thehalf-life of biological molecules. By way of example only, one may wishto modify the termini by acylation or amination.

[0458] Moderate conjugation-type modifications for use with the presentinvention include incorporating a salvage receptor binding epitope intothe antibody fragment. Techniques for achieving this include mutation ofthe appropriate region of the antibody fragment or incorporating theepitope as a peptide tag that is attached to the antibody fragment. WO96/32478 is specifically incorporated herein by reference for thepurposes of further exemplifying such technology. Salvage receptorbinding epitopes are typically regions of three or more amino acids fromone or two lops of the Fc domain that are transferred to the analogousposition on the antibody fragment. The salvage receptor binding epitopesof WO 98/45331 are incorporated herein by reference for use with thepresent invention.

[0459] F. Immunoconjugates Binding to Anionic Phospholipids andAminophospholipids

[0460] The present inventors earlier developed a range ofimmunoconjugates that bind to aminophospholipids for use in targetingtumor vasculature (U.S. Pat. No. 6,312,694, specifically incorporatedherein by reference). These agents use aminophospholipid-bindingproteins, such as annexins and kininogens, and antibodies againstaminophospholipids, such as PS and PE, to deliver attached therapeuticagents to tumor and intratumoral vasculature. The present invention nowprovides selected anti-PS antibodies with improved properties, such as3G4 (ATCC 4545) and 9D2, and these and competing antibodies can now alsobe used as the antibody portions of immunoconjugates.

[0461] In addition to the use of vascular targeting agents that bind toaminophospholipids (U.S. Pat. No. 6,312,694), the present discovery thatanionic phospholipids, as well as aminophospholipids, are stable andtargetable entities within tumor vasculature provides for the use of arange of new tumor vascular targeting agents. The new compounds, notsuggested in the earlier work directed to aminophospholipids, useantibodies directed against anionic phospholipids to deliver toxins,cytokines, coagulants and other therapeutic agents to anionicphospholipids upregulated on tumor and intratumoral vasculature. Asdetailed above in regard to the naked antibodies, the development ofthese aspects of the invention required the generation of biologicaltools, particularly antibodies, with exquisite specificity for differentphospholipids, anionic phospholipids and aminophospholipids.

[0462] As the present invention shows that anionic phospholipids andaminophospholipids, such as PS, PE, PI, PA and PG, and most particularlyPS and PE, are safe and effective targets for anti-viral therapy,antibodies and peptides that bind to these components, particularly PSand PE, may now be advantageously linked to a range of known anti-viralagents. These anti-viral conjugates include both peptide-based andantibody-based conjugates, the latter of which may be termed anti-viralimmunoconjugates or “immunovirocides”.

[0463] In these aspects of the invention, any antibody against ananionic phospholipid can be used to prepare an immunoconjugate,immunotoxin or coaguligand, with antibodies such as the secondgeneration antibodies, particularly 9D2-like and 3G4-like antibodies,with their advantageous anionic phospholipid binding profiles, beingpreferred. Agents for use in such immunoconjugates preferably includeanti-cellular or cytotoxic agents, coagulants (coagulation factors),cytokines, radiotherapeutic agents, anti-angiogenic agents,apoptosis-inducing agents, anti-tubulin drugs and anti-viral agents (andthe PE-binding peptides, such as duramycin derivatives, as disclosed indetail herein). In the anti-viral immunoconjugates, there is norequirement to use a second generation antibody as disclosed herein,although these can certainly be employed. Any antibody toaminophospholipids or anionic phospholipids may be thus be linked to ananti-viral agent to form an anti-viral immunoconjugates orimmunovirocide in accordance with the present invention.

[0464] F1. Anti-Cellular and Cytotoxic Agents

[0465] For certain applications, the therapeutic agents will becytotoxic or pharmacological agents, particularly cytotoxic, cytostaticor otherwise anti-cellular agents having the ability to kill or suppressthe growth or cell division of cells, particularly tumor endothelialcells or tumor cells. In general, these aspects of the inventioncontemplate the use of any pharmacological agent that can be conjugatedto an antibody against an anionic phospholipid, preferably a 9D2-basedor 3G4-based antibody, and delivered in active form to the targetedendothelium.

[0466] Exemplary anti-cellular agents include chemotherapeutic agents,as well as cytotoxins. Chemotherapeutic agents that may be used include:hormones, such as steroids; anti-metabolites, such as cytosinearabinoside, fluorouracil, methotrexate or aminopterin; anthracyclines;mitomycin C; vinca alkaloids; demecolcine; etoposide; mithramycin;anti-tumor alkylating agents, such as chlorambucil or melphalan. Otherembodiments may include agents such as cytokines. Basically, anyanti-cellular agent may be used, so long as it can be successfullyconjugated to, or associated with, an antibody in a manner that willallow its targeting, internalization, release and/or presentation toblood components at the site of the targeted cells, such as endothelialcells.

[0467] There may be circumstances, such as when the target antigen doesnot internalize by a route consistent with efficient intoxication by thetoxic compound, where one will desire to target chemotherapeutic agents,such as anti-tumor drugs, cytokines, antimetabolites, alkylating agents,hormones, and the like. A variety of chemotherapeutic and otherpharmacological agents have now been successfully conjugated toantibodies and shown to function pharmacologically, includingdoxorubicin, daunomycin, methotrexate, vinblastine, neocarzinostatin,macromycin, trenimon and α-amanitin.

[0468] In other circumstances, any potential side-effects fromcytotoxin-based therapy may be eliminated by the use of DNA synthesisinhibitors, such as daunorubicin, doxorubicin, adriamycin, and the like.These agents are therefore preferred examples of anti-cellular agentsfor use in certain aspects of the present invention. In terms ofcytostatic agents, such compounds generally disturb the natural cellcycle of a target cell, preferably so that the cell is taken out of thecell cycle.

[0469] A wide variety of cytotoxic agents are known that may beconjugated to an antibody against an anionic phospholipid, preferably a9D2-based or 3G4-based antibody. Examples include numerous usefulplant-, fungus- or bacteria-derived toxins, which, by way of example,include various A chain toxins, particularly ricin A chain; ribosomeinactivating proteins, such as saporin or gelonin; α-sarcin;aspergillin; restrictocin; ribonucleases, such as placentalribonuclease; diphtheria toxin; and pseudomonas exotoxin, to name just afew.

[0470] Of the toxins, the use of gelonin and ricin A chains arepreferred. The use of gelonin as the effector or toxin portion ofimmunoconjugates that bind to markers expressed, accessible to binding,adsorbed or localized on intratumoral blood vessels of a vascularizedtumor is described in U.S. Pat. No. 6,051,230, specifically incorporatedherein by reference, and in U.S. Pat. No. 6,451,312, which particularlyconcerns gelonin linked to VEGF as a targeting agent.

[0471] As to ricin A chains, a further preferred toxin moiety is toxin Achain that has been treated to modify or remove carbohydrate residues,so-called deglycosylated A chain (dgA). Deglycosylated ricin A chain ispreferred because of its extreme potency, longer half-life, and becauseit is economically feasible to manufacture it in a clinical grade andscale.

[0472] It may be desirable from a pharmacological standpoint to employthe smallest molecule possible that nevertheless provides an appropriatebiological response. One may thus desire to employ smaller A chainpeptides that will provide an adequate anti-cellular response. To thisend, it has been discovered that ricin A chain may be “truncated” by theremoval of 30 N-terminal amino acids by Nagarase (Sigma), and stillretain an adequate toxin activity. It is proposed that where desired,this truncated A chain may be employed in conjugates in accordance withthe invention.

[0473] Alternatively, one may find that the application of recombinantDNA technology to the toxin A chain moiety will provide additionalbenefits in accordance the invention. In that the cloning and expressionof biologically active ricin A chain has been achieved, it is nowpossible to identify and prepare smaller, or otherwise variant peptides,which nevertheless exhibit an appropriate toxin activity. Moreover, thefact that ricin A chain has now been cloned allows the application ofsite-directed mutagenesis, through which one can readily prepare andscreen for A chain-derived peptides and obtain additional usefulmoieties for use in connection with the present invention.

[0474] F2. Cytokines

[0475] Cytokines and chemokines are particular examples of agents forlinking to the antibodies of the present invention. A range of cytokinesmay be used, including IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-11, IL-13,TGF-β, M-CSF, G-CSF, TNFβ, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM,TMF, IFN-α, IFN-β. More preferred cytokines include IL-1α, IL-1β, IL-2,IL-6, IL-10, GM-CSF, IFNγ, monocyte chemoattractant protein-1 (MCP-1),platelet-derived growth factor-BB (PDGF-BB) and C-reactive protein (CRP)and the like. Particularly preferred examples are TNFα, TNFα inducersand IL-12.

[0476] TNFα increases vascular permeability. This agent is contemplatedfor attachment to an antibody of the invention, particularly where theresultant immunoconjugate is used in combination therapy for thetreatment of cancer. The antibody will deliver the attached TNFα to thetumor environment, and the enhanced vascular permeability cause in thetumor will facilitate the penetration of a second anti-cancer agent intothe tumor, thus amplifying the overall anti-tumor effect. scFvconstructs are particularly contemplated for use in such embodiments.This is partly because TNFα functions as a trimer and the scFvconstructs will be able to trimerize readily.

[0477] IL-12, for example, may be attached to an antibody and used toredirect host defenses to attack the tumor vessels. In using IL-12, anscFv form of antigen binding region may be preferred. The chemokine LEC(liver-expressed chemokine, also known as NCC-4, HCC-4, or LMC) isanother preferred component (Giovarelli et al., 2000). LEC ischemotactic for dendritic cells, monocytes, T cells, NK cells andneutrophils and can therefore improve host-mediated anti-tumorresponses.

[0478] F3. Coagulation Factors

[0479] An antibody against an anionic phospholipid, or a secondgeneration antibody based upon the preferred 9D2 and 3G4 (ATCC 4545)antibodies of the invention, may be linked to a component that iscapable of directly or indirectly stimulating coagulation, to form acoaguligand. U.S. Pat. Nos. 6,093,399, 6,004,555, 5,877,289 and6,036,955 are specifically incorporated herein by reference for purposesof further describing the operative association of coagulants withantibodies to form coaguligands.

[0480] The antibodies of the invention may be directly linked to thecoagulant or coagulation factor, or may be linked to a second bindingregion that binds and then releases the coagulant or coagulation factor.As used herein, the terms “coagulant” and “coagulation factor” are eachused to refer to a component that is capable of directly or indirectlystimulating coagulation under appropriate conditions, preferably whenprovided to a specific in vivo environment, such as the tumorvasculature.

[0481] Preferred coagulation factors are Tissue Factor compositions,such as truncated TF (tTF), dimeric, multimeric and mutant TF molecules.“Truncated TF” (tTF) refers to TF constructs that are renderedmembrane-binding deficient by removal of sufficient amino acid sequencesto effect this change in property. A “sufficient amount” in this contextis an amount of transmembrane amino acid sequence originally sufficientto enter the TF molecule in the membrane, or otherwise mediatefunctional membrane binding of the TF protein. The removal of such a“sufficient amount of transmembrane spanning sequence” therefore createsa truncated Tissue Factor protein or polypeptide deficient inphospholipid membrane binding capacity, such that the protein issubstantially a soluble protein that does not significantly bind tophospholipid membranes. Truncated TF thus substantially fails to convertFactor VII to Factor VIIa in a standard TF assay, and yet retainsso-called catalytic activity including activating Factor X in thepresence of Factor VIIa.

[0482] U.S. Pat. Nos. 5,504,067, 6,156,321, 6,132,729 and 6,132,730 arespecifically incorporated herein by reference for the purposes offurther describing such truncated Tissue Factor proteins. Preferably,the Tissue Factors for use in these aspects of the present inventionwill generally lack the transmembrane and cytosolic regions (amino acids220-263) of the protein. However, there is no need for the truncated TFmolecules to be limited to molecules of the exact length of 219 aminoacids.

[0483] Tissue Factor compositions may also be useful as dimers. Any ofthe truncated, mutated or other Tissue Factor constructs may be preparedin a dimeric form for use in the present invention. As will be known tothose of ordinary skill in the art, such TF dimers may be prepared byemploying the standard techniques of molecular biology and recombinantexpression, in which two coding regions are prepared in-frame andexpressed from an expression vector. Equally, various chemicalconjugation technologies may be employed in connection with thepreparation of TF dimers. The individual TF monomers may be derivatizedprior to conjugation. All such techniques would be readily known tothose of skill in the art.

[0484] If desired, the Tissue Factor dimers or multimers may be joinedvia a biologically-releasable bond, such as a selectively-cleavablelinker or amino acid sequence. For example, peptide linkers that includea cleavage site for an enzyme preferentially located or active within atumor environment are contemplated. Exemplary forms of such peptidelinkers are those that are cleaved by urokinase, plasmin, thrombin,Factor IXa, Factor Xa, or a metalloproteinase, such as collagenase,gelatinase or stromelysin.

[0485] In certain embodiments, the Tissue Factor dimers may furthercomprise a hindered hydrophobic membrane insertion moiety, to laterencourage the functional association of the Tissue Factor with thephospholipid membrane, but only under certain defined conditions. Asdescribed in the context of the truncated Tissue Factors, hydrophobicmembrane-association sequences are generally stretches of amino acidsthat promote association with the phospholipid environment due to theirhydrophobic nature. Equally, fatty acids may be used to provide thepotential membrane insertion moiety.

[0486] Such membrane insertion sequences may be located either at theN-terminus or the C-terminus of the TF molecule, or generally appendedat any other point of the molecule so long as their attachment theretodoes not hinder the functional properties of the TF construct. Theintent of the hindered insertion moiety is that it remainsnon-functional until the TF construct localizes within the tumorenvironment, and allows the hydrophobic appendage to become accessibleand even further promote physical association with the membrane. Again,it is contemplated that biologically-releasable bonds andselectively-cleavable sequences will be particularly useful in thisregard, with the bond or sequence only being cleaved or otherwisemodified upon localization within the tumor environment and exposure toparticular enzymes or other bioactive molecules.

[0487] In other embodiments, the tTF constructs may be multimeric orpolymeric. In this context a “polymeric construct” contains 3 or moreTissue Factor constructs. A “multimeric or polymeric TF construct” is aconstruct that comprises a first TF molecule or derivative operativelyattached to at least a second and a third TF molecule or derivative. Themultimers may comprise between about 3 and about 20 such TF molecules.The individual TF units within the multimers or polymers may also belinked by selectively-cleavable peptide linkers or otherbiological-releasable bonds as desired. Again, as with the TF dimersdiscussed above, the constructs may be readily made using eitherrecombinant manipulation and expression or using standard syntheticchemistry.

[0488] Even further TF constructs useful in context of the presentinvention are those mutants deficient in the ability to activate FactorVII. Such “Factor VII activation mutants” are generally defined hereinas TF mutants that bind functional Factor VII/VIIa, proteolyticallyactivate Factor X, but are substantially free from the ability toproteolytically activate Factor VII. Accordingly, such constructs are TFmutants that lack Factor VII activation activity.

[0489] The ability of such Factor VII activation mutants to function inpromoting tumor-specific coagulation is based upon their specificdelivery to the tumor vasculature, and the presence of Factor VIIa atlow levels in plasma. Upon administration of such a Factor VIIactivation mutant conjugate, the mutant will be localized within thevasculature of a vascularized tumor. Prior to localization, the TFmutant would be generally unable to promote coagulation in any otherbody sites, on the basis of its inability to convert Factor VII toFactor VIIa. However, upon localization and accumulation within thetumor region, the mutant will then encounter sufficient Factor VIIa fromthe plasma in order to initiate the extrinsic coagulation pathway,leading to tumor-specific thrombosis. Exogenous Factor VIIa could alsobe administered to the patient.

[0490] Any one or more of a variety of Factor VII activation mutants maybe prepared and used in connection with the present invention. There isa significant amount of scientific knowledge concerning the recognitionsites on the TF molecule for Factor VII/VIIa. It will thus be understoodthat the Factor VII activation region generally lies between about aminoacid 157 and about amino acid 167 of the TF molecule. However, it iscontemplated that residues outside this region may also prove to berelevant to the Factor VII activating activity, and one may thereforeconsider introducing mutations into any one or more of the residuesgenerally located between about amino acid 106 and about amino acid 209of the TF sequence (WO 94/07515; WO 94/28017; each incorporated hereinby reference).

[0491] As detailed in U.S. Pat. Nos. 6,093,399, 6,004,555, 5,877,289 and6,036,955, a variety of other coagulation factors may be used inconnection with the present invention, as exemplified by the agents setforth below. Thrombin, Factor V/Va and derivatives, Factor VIII/VIIIaand derivatives, Factor IX/IXa and derivatives, Factor X/Xa andderivatives, Factor XI/XIa and derivatives, Factor XII/XIIa andderivatives, Factor XIII/XIIIa and derivatives, Factor X activator andFactor V activator may be used in the present invention.

[0492] Russell's viper venom Factor X activator is contemplated for usein this invention. Monoclonal antibodies specific for the Factor Xactivator present in Russell's viper venom have also been produced, andcould be used to specifically deliver the agent as part of a bispecificbinding ligand.

[0493] Thromboxane A₂ is formed from endoperoxides by the sequentialactions of the enzymes cyclooxygenase and thromboxane synthetase inplatelet microsomes. Thromboxane A₂ is readily generated by plateletsand is a potent vasoconstrictor, by virtue of its capacity to produceplatelet aggregation. Both thromboxane A₂ and active analogues thereofare contemplated for use in the present invention.

[0494] Thromboxane synthase, and other enzymes that synthesizeplatelet-activating prostaglandins, may also be used as “coagulants” inthe present context. Monoclonal antibodies to, and immunoaffinitypurification of, thromboxane synthase are known; as is the cDNA forhuman thromboxane synthase.

[0495] α2-antiplasmin, or α2-plasmin inhibitor, is a proteinaseinhibitor naturally present in human plasma that functions toefficiently inhibit the lysis of fibrin clots induced by plasminogenactivator. α2-antiplasmin is a particularly potent inhibitor, and iscontemplated for use in the present invention.

[0496] As the cDNA sequence for α2-antiplasmin is available, recombinantexpression and/or fusion proteins are preferred. Monoclonal antibodiesagainst α2-antiplasmin are also available that may be used in thebispecific binding ligand embodiments of the invention. These antibodiescould both be used to deliver exogenous α2-antiplasmin to the targetsite or to garner endogenous α2-antiplasmin and concentrate it withinthe targeted region.

[0497] F4. Anti-Tubulin Drugs

[0498] A range of drugs exert their effects via interfering with tubulinactivity. As tubulin functions are essential to mitosis and cellviability, certain “anti-tubulin drugs” are powerful chemotherapeuticagents. “Anti-tubulin drug(s)”, as used herein, means any agent, drug,prodrug or combination thereof that inhibits cell mitosis, preferably bydirectly or indirectly inhibiting tubulin activities necessary for cellmitosis, preferably tubulin polymerization or depolymerization.

[0499] Some of the more well known and currently preferred anti-tubulindrugs for use with the present invention are colchicine; taxanes, suchas taxol; vinca alkaloids, such as vinblastine, vincristine andvindescine; and combretastatins. Other suitable anti-tubulin drugs arecytochalasins (including B, J, E), dolastatin, auristatin PE,paclitaxel, ustiloxin D, rhizoxin, 1069C85, colcemid, albendazole,azatoxin and nocodazole.

[0500] As described in U.S. Pat. Nos. 5,892,069, 5,504,074 and5,661,143, each specifically incorporated herein by reference,combretastatins are estradiol derivatives that generally inhibit cellmitosis. Exemplary combretastatins that may be used in conjunction withthe invention include those based upon combretastatin A, B and/or D andthose described in U.S. Pat. Nos. 5,892,069, 5,504,074 and 5,661,143.Combretastatins A-1, A-2, A-3, A-4, A-5, A-6, B-1, B-2, B-3 and B-4 areexemplary of the foregoing types.

[0501] U.S. Pat. Nos. 5,569,786 and 5,409,953, are incorporated hereinby reference for purposes of describing the isolation, structuralcharacterization and synthesis of each of combretastatin A-1, A2, A-3,B-1, B-2, B-3 and B-4 and formulations and methods of using suchcombretastatins to treat neoplastic growth. Any one or more of suchcombretastatins may be used in conjunction with the present invention.

[0502] Combretastatin A-4, as described in U.S. Pat. Nos. 5,892,069,5,504,074, 5,661,143 and 4,996,237, each specifically incorporatedherein by reference, may also be used herewith. U.S. Pat. No. 5,561,122is further incorporated herein by reference for describing suitablecombretastatin A-4 prodrugs, which are contemplated for combined usewith the present invention.

[0503] U.S. Pat. No. 4,940,726, specifically incorporated herein byreference, particularly describes macrocyclic lactones denominatedcombretastatin D-1 and ‘Combretastatin D-2’, each of which may be usedin combination with the compositions and methods of the presentinvention. U.S. Pat. No. 5,430,062, specifically incorporated herein byreference, concerns stilbene derivatives and combretastatin analogueswith anti-cancer activity that may be used in combination with thepresent invention.

[0504] F5. Anti-Angiogenic Agents

[0505] Anti-angiogenic agents are useful for attachment to theantibodies and peptides of the invention. Many anti-cancer agents havean anti-angiogenic effect as part of their mechanism of action. Any oneor more of such agents described for use in combination therapies,including those in Table E, may also be conjugated to an antibody of theinvention, as described herein. Certain other agents have beendiscovered, designed or selected to have an anti-angiogenic effect as aprimary mechanism of action. Examples of such agents are describedbelow, any of which may also be used to prepare an immunoconjugate orused separately in combination therapy with the invention.

[0506] Numerous tyrosine kinase inhibitors useful for the treatment ofangiogenesis, as manifest in various diseases states, are now known.These include, for example, the 4-aminopyrrolo[2,3-d]pyrimidines of U.S.Pat. No. 5,639,757, specifically incorporated herein by reference, whichmay also be used in combination with the present invention. Furtherexamples of organic molecules capable of modulating tyrosine kinasesignal transduction via the VEGFR2 receptor are the quinazolinecompounds and compositions of U.S. Pat. No. 5,792,771, which isspecifically incorporated herein by reference for the purpose ofdescribing further combinations for use with the present invention inthe treatment of angiogenic diseases.

[0507] Compounds of other chemical classes have also been shown toinhibit angiogenesis and may be used in combination with the presentinvention. For example, steroids such as the angiostatic4,9(11)-steroids and C21-oxygenated steroids, as described in U.S. Pat.No. 5,972,922, specifically incorporated herein by reference, may beemployed in combined therapy. U.S. Pat. Nos. 5,712,291 and 5,593,990,each specifically incorporated herein by reference, describe thalidomideand related compounds, precursors, analogs, metabolites and hydrolysisproducts, which may also be used in combination with the presentinvention to inhibit angiogenesis. The compounds in U.S. Pat. Nos.5,712,291 and 5,593,990 can be administered orally. Further exemplaryanti-angiogenic agents that are useful in connection with combinedtherapy are listed in Table B. Each of the agents listed therein areexemplary and by no means limiting. TABLE B Inhibitors and NegativeRegulators of Angiogenesis Substances References Angiostatin O'Reilly etal., 1994 Endostatin O'Reilly et al., 1997 16 kDa prolactin fragmentFerrara et al., 1991; Clapp et al., 1993; D'Angelo et al., 1995; Lee etal., 1998 Laminin peptides Kleinman et al., 1993; Yamamura et al., 1993;Iwamoto et al., 1996; Tryggvason, 1993 Fibronectin peptides Grant etal., 1998; Sheu et al., 1997 Tissue metalloproteinase inhibitors Sang,1998 (TIMP 1, 2, 3, 4) Plasminogen activator inhibitors Soff et al.,1995 (PAI-1, -2) Tumor necrosis factor α (high dose, in Frater-Schroderet al., 1987 vitro) TGF-β1 RayChadhury and D'Amore, 1991; Tada et al.,1994 Interferons (IFN-α, -β, γ) Moore et al., 1998; Lingen et al., 1998ELR- CXC Chemokines: Moore et al., 1998; Hiscox and Jiang, 1997;Coughlin IL-12; SDF-1; MIG; Platelet factor 4 et al., 1998; Tanaka etal., 1997 (PF-4); IP-10 Thrombospondin (TSP) Good et al., 1990; Frazier,1991; Bornstein, 1992; Tolsma et al., 1993; Sheibani and Frazier, 1995;Volpert et al., 1998 SPARC Hasselaar and Sage, 1992; Lane et al., 1992;Jendraschak and Sage, 1996 2-Methoxyoestradiol Fotsis et al., 1994Proliferin-related protein Jackson et al., 1994 Suramin Gagliardi etal., 1992; Takano et al., 1994; Waltenberger et al., 1996; Gagliardi etal., 1998; Manetti et al., 1998 Thalidomide D'Amato et al., 1994; Kenyonet al., 1997 Wells, 1998 Cortisone Thorpe et al., 1993 Folkman et al.,1983 Sakamoto et al., 1986 Linomide Vukanovic et al., 1993; Ziche etal., 1998; Nagler et al., 1998 Fumagillin (AGM-1470; TNP-470) Sipos etal., 1994; Yoshida et al., 1998 Tamoxifen Gagliardi and Collins, 1993;Linder and Borden, 1997; Haran et al., 1994 Korean mistletoe extractYoon et al., 1995 (Viscum album coloratum) Retinoids Oikawa et al.,1989; Lingen et al., 1996; Majewski et al. 1996 CM101 Hellerqvist etal., 1993; Quinn et al., 1995; Wamil et al., 1997; DeVore et al., 1997Dexamethasone Hori et al., 1996; Wolff et al., 1997 Leukemia inhibitoryfactor (LIF) Pepper et al., 1995

[0508] Certain preferred components for use in inhibiting angiogenesisare angiostatin, endostatin, vasculostatin, canstatin and maspin. Theprotein named “angiostatin” is disclosed in U.S. Pat. Nos. 5,776,704;5,639,725 and 5,733,876, each incorporated herein by reference.Angiostatin is a protein having a molecular weight of between about 38kD and about 45 kD, as determined by reducing polyacrylamide gelelectrophoresis, which contains approximately Kringle regions 1 through4 of a plasminogen molecule. Angiostatin generally has an amino acidsequence substantially similar to that of a fragment of murineplasminogen beginning at amino acid number 98 of an intact murineplasminogen molecule.

[0509] The amino acid sequence of angiostatin varies slightly betweenspecies. For example, in human angiostatin, the amino acid sequence issubstantially similar to the sequence of the above described murineplasminogen fragment, although an active human angiostatin sequence maystart at either amino acid number 97 or 99 of an intact humanplasminogen amino acid sequence. Further, human plasminogen may be used,as it has similar anti-angiogenic activity, as shown in a mouse tumormodel.

[0510] Certain anti-angiogenic therapies have already been shown tocause tumor regressions, and angiostatin is one such agent. Endostatin,a 20 kDa COOH-terminal fragment of collagen XVIII, the bacterialpolysaccharide CM101, and the antibody LM609 also have angiostaticactivity. However, in light of their other properties, they are referredto as anti-vascular therapies or tumor vessel toxins, as they not onlyinhibit angiogenesis but also initiate the destruction of tumor vesselsthrough mostly undefined mechanisms.

[0511] Angiostatin and endostatin have become the focus of intensestudy, as they are the first angiogenesis inhibitors that havedemonstrated the ability to not only inhibit tumor growth but also causetumor regressions in mice. There are multiple proteases that have beenshown to produce angiostatin from plasminogen including elastase,macrophage metalloelastase (MME), matrilysin (MMP-7), and 92 kDagelatinase B/type IV collagenase (MMP-9).

[0512] MME can produce angiostatin from plasminogen in tumors andgranulocyte-macrophage colony-stimulating factor (GMCSF) upregulates theexpression of MME by macrophages inducing the production of angiostatin.The role of MME in angiostatin generation is supported by the findingthat MME is in fact expressed in clinical samples of hepatocellularcarcinomas from patients. Another protease thought to be capable ofproducing angiostatin is stromelysin-1 (MMP-3). MMP-3 has been shown toproduce angiostatin-like fragments from plasminogen in vitro. Themechanism of action for angiostatin is currently unclear, it ishypothesized that it binds to an unidentified cell surface receptor onendothelial cells inducing endothelial cell to undergo programmed celldeath or mitotic arrest.

[0513] Endostatin appears to be an even more powerful anti-angiogenesisand anti-tumor agent although its biology is less clear. Endostatin iseffective at causing regressions in a number of tumor models in mice.Tumors do not develop resistance to endostatin and, after multiplecycles of treatment, tumors enter a dormant state during which they donot increase in volume. In this dormant state, the percentage of tumorcells undergoing apoptosis was increased, yielding a population thatessentially stays the same size. Endostatin is thought to bind anunidentified endothelial cell surface receptor that mediates its effect.

[0514] U.S. Pat. No. 5,854,205, to Folkman and O'Reilly, specificallyincorporated herein by reference, concerns endostatin and its use as aninhibitor of endothelial cell proliferation and angiogenesis. Theendostatin protein corresponds to a C-terminal fragment of collagen typeXVIII, and the protein can be isolated from a variety of sources. U.S.Pat. No. 5,854,205 also teaches that endostatin can have an amino acidsequence of a fragment of collagen type XVIII, a collasen type XV, orBOVMPE 1 pregastric esterase. Combinations of endostatin with otheranti-angiogenic proteins, particularly angiostatin, are also describedby U.S. Pat. No. 5,854,205, such that the combined compositions arecapable of effectively regressing the mass of an angiogenesis-dependenttumor.

[0515] CM101 is a bacterial polysaccharide that has been wellcharacterized in its ability to induce neovascular inflammation intumors. CM101 binds to and cross-links receptors expressed ondedifferentiated endothelium that stimulates the activation of thecomplement system. It also initiates a cytokine-driven inflammatoryresponse that selectively targets the tumor. It is a uniquelyantipathoangiogenic agent that downregulates the expression VEGF and itsreceptors. CM101 is currently in clinical trials as an anti-cancer drug,and can be used in combination with this invention.

[0516] Thrombospondin (TSP-1) and platelet factor 4 (PF4) may also beused in the present invention. These are both angiogenesis inhibitorsthat associate with heparin and are found in platelet α-granules. TSP-1is a large 450 kDa multi-domain glycoprotein that is constituent of theextracellular matrix. TSP-1 binds to many of the proteoglycan moleculesfound in the extracellular matrix including, HSPGs, fibronectin,laminin, and different types of collagen. TSP-1 inhibits endothelialcell migration and proliferation in vitro and angiogenesis in vivo.TSP-1 can also suppress the malignant phenotype and tumorigenesis oftransformed endothelial cells. The tumor suppressor gene p53 has beenshown to directly regulate the expression of TSP-1 such that, loss ofp53 activity causes a dramatic reduction in TSP-1 production and aconcomitant increase in tumor initiated angiogenesis.

[0517] PF4 is a 70aa protein that is member of the CXC ELR-family ofchemokines that is able to potently inhibit endothelial cellproliferation in vitro and angiogenesis in vivo. PF4 administeredintratumorally or delivered by an adenoviral vector is able to cause aninhibition of tumor growth.

[0518] Interferons and metalloproteinase inhibitors are two otherclasses of naturally occurring angiogenic inhibitors that can bedelivered according to the present invention. The anti-endothelialactivity of the interferons has been known since the early 1980s,however, the mechanism of inhibition is still unclear. It is known thatthey can inhibit endothelial cell migration and that they do have someanti-angiogenic activity in vivo that is possibly mediated by an abilityto inhibit the production of angiogenic promoters by tumor cells.Vascular tumors in particular are sensitive to interferon, for example,proliferating hemangiomas can be successfully treated with IFNα.

[0519] Tissue inhibitors of metalloproteinases (TIMPs) are a family ofnaturally occurring inhibitors of matrix metalloproteases (MMPs) thatcan also inhibit angiogenesis and can be used in the treatment protocolsof the present invention. MMPs play a key role in the angiogenic processas they degrade the matrix through which endothelial cells andfibroblasts migrate when extending or remodeling the vascular network.In fact, one member of the MMPs, MMP-2, has been shown to associate withactivated endothelium through the integrin αvβ3 presumably for thispurpose. If this interaction is disrupted by a fragment of MMP-2, thenangiogenesis is downregulated and in tumors growth is inhibited.

[0520] There are a number of pharmacological agents that inhibitangiogenesis, any one or more of which may be used as part of thepresent invention. These include AGM-1470/TNP-470, thalidomide, andcarboxyamidotriazole (CAI). Fumagillin was found to be a potentinhibitor of angiogenesis in 1990, and since then the syntheticanalogues of fumagillin, AGM-1470 and TNP-470 have been developed. Bothof these drugs inhibit endothelial cell proliferation in vitro andangiogenesis in vivo. TNP-470 has been studied extensively in humanclinical trials with data suggesting that long-term administration isoptimal.

[0521] Thalidomide was originally used as a sedative but was found to bea potent teratogen and was discontinued. In 1994 it was found thatthalidomide is an angiogenesis inhibitor. Thalidomide is currently inclinical trials as an anti-cancer agent as well as a treatment ofvascular eye diseases.

[0522] CAI is a small molecular weight synthetic inhibitor ofangiogenesis that acts as a calcium channel blocker that prevents actinreorganization, endothelial cell migration and spreading on collagen IV.CAI inhibits neovascularization at physiological attainableconcentrations and is well tolerated orally by cancer patients. Clinicaltrials with CAI have yielded disease stabilization in 49% of cancerpatients having progressive disease before treatment.

[0523] Cortisone in the presence of heparin or heparin fragments wasshown to inhibit tumor growth in mice by blocking endothelial cellproliferation. The mechanism involved in the additive inhibitory effectof the steroid and heparin is unclear although it is thought that theheparin may increase the uptake of the steroid by endothelial cells. Themixture has been shown to increase the dissolution of the basementmembrane underneath newly formed capillaries and this is also a possibleexplanation for the additive angiostatic effect. Heparin-cortisolconjugates also have potent angiostatic and anti-tumor effects activityin vivo.

[0524] Further specific angiogenesis inhibitors may be delivered totumors using the tumor targeting methods of the present invention. Theseinclude, but are not limited to, Anti-Invasive Factor, retinoic acidsand paclitaxel (U.S. Pat. No. 5,716,981; incorporated herein byreference); AGM-1470 (Ingber et al., 1990; incorporated herein byreference); shark cartilage extract (U.S. Pat. No. 5,618,925;incorporated herein by reference); anionic polyamide or polyureaoligomers (U.S. Pat. No. 5,593,664; incorporated herein by reference);oxindole derivatives (U.S. Pat. No. 5,576,330; incorporated herein byreference); estradiol derivatives (U.S. Pat. No. 5,504,074; incorporatedherein by reference); and thiazolopyrimidine derivatives (U.S. Pat. No.5,599,813; incorporated herein by reference) are also contemplated foruse as anti-angiogenic compositions for the combined uses of the presentinvention.

[0525] Compositions comprising an antagonist of an α_(v)β₃ integrin mayalso be used to inhibit angiogenesis as part of the present invention.As disclosed in U.S. Pat. No. 5,766,591 (incorporated herein byreference), RGD-containing polypeptides and salts thereof, includingcyclic polypeptides, are suitable examples of α_(v)β₃ integrinantagonists.

[0526] As angiopoietins are ligands for Tie2, other methods oftherapeutic intervention based upon altering signaling through the Tie2receptor can also be used in combination herewith. For example, asoluble Tie2 receptor capable of blocking Tie2 activation (Lin et al.,1998a) can be employed. Delivery of such a construct using recombinantadenoviral gene therapy has been shown to be effective in treatingcancer and reducing metastases (Lin et al., 1998a).

[0527] The angiopoietins, in common with the members of the VEGF family,are growth factors specific for vascular endothelium (Davis andYancopoulos, 1999; Holash et al., 1999; incorporated herein byreference). The angiopoietins first described were a naturally occurringreceptor activator or agonist, angiopoietin-1 (Ang-1), and a naturallyoccurring receptor antagonist, angiopoietin-2 (Ang-2), both of which actby means of the endothelial cell tyrosine kinase receptor, Tie2.

[0528] Two new angiopoietins, angiopoietin-3 (mouse) and angiopoietin-4(human) have also been identified (Valenzuela et al., 1999).Angiopoietin-3 appears to act as an antagonist (like Ang-2), whereasangiopoietin-4 appears to function as an agonist (like Ang-1)(Valenzuela et al., 1999). A protein termed angiopoietin-3 was alsocloned from human heart and reported not to have mitogenic effects onendothelial cells (Kim et al., 1999).

[0529] Whereas VEGF is necessary for the early stages of vasculardevelopment, angiopoietin-1 is generally required for the later stagesof vascularization. VEGF thus acts to promote endothelial celldifferentiation, proliferation and primitive vessel formation.

[0530] Angiopoietin-1 acts, via the Tie2 receptor, to promotemaintenance and stabilization of mature vessels. Angiopoietin-1 is thusa maturation or stabilization factor, thought to convert immaturevessels to immature vessels by promoting interactions betweenendothelial cells and surrounding support cells (Holash et al., 1999).

[0531] F6. Apoptosis-Inducing Agents

[0532] The present invention may also be used to deliver agents thatinduce apoptosis in any cells within the tumor, including tumor cellsand tumor vascular endothelial cells. Many anticancer agents have, aspart of their mechanism of action, an apoptosis-inducing effect. Any oneor more of such agents described for use in combination therapies,including those in Table F, may also be conjugated to an antibody of theinvention, as described herein. Certain other agents have beendiscovered, designed or selected to have an apoptosis-inducing effect asa primary mechanism. Examples of such agents are described below, any ofwhich may also be used to prepare an immunoconjugate or used separatelyin combination therapy with the invention.

[0533] Many forms of cancer have reports of mutations in tumorsuppressor genes, such as p53. Inactivation of p53 results in a failureto promote apoptosis. With this failure, cancer cells progress intumorigenesis, rather than become destined for cell death. Thus,delivery of tumor suppressors is also contemplated for use in thepresent invention to stimulate cell death. Exemplary tumor suppressorsinclude, but are not limited to, p53, Retinoblastoma gene (Rb), Wilm'stumor (WT1), bax alpha, interleukin-1b-converting enzyme and family,MEN-1 gene, neurofibromatosis, type 1 (NF1), cdk inhibitor p16,colorectal cancer gene (DCC), familial adenomatosis polyposis gene(FAP), multiple tumor suppressor gene (MTS-1), BRCA1 and BRCA2.

[0534] Preferred for use are the p53 (U.S. Pat. Nos. 5,747,469;5,677,178; and 5,756,455; each incorporated herein by reference),Retinoblastoma, BRCA1 (U.S. Pat. Nos. 5,750,400; 5,654,155; 5,710,001;5,756,294; 5,709,999; 5,693,473; 5,753,441; 5,622,829; and 5,747,282;each incorporated herein by reference), MEN-1 (GenBank accession numberU93236) and adenovirus E1A (U.S. Pat. No. 5,776,743; incorporated hereinby reference) genes.

[0535] Other oncogenes that inhibit apoptosis or programmed cell deathinclude, but are not limited to, bcr-abl, bcl-2 (distinct from bcl-1,cyclin D1; GenBank accession numbers M14745, X06487; U.S. Pat. Nos.5,650,491; and 5,539,094; each incorporated herein by reference) andfamily members including Bcl-xl, Mcl-1, Bak, A1, A20. Overexpression ofbcl-2 was first discovered in T cell lymphomas. bcl-2 functions as anoncogene by binding and inactivating Bax, a protein in the apoptoticpathway. Inhibition of bcl-2 function prevents inactivation of Bax, andallows the apoptotic pathway to proceed. Thus, inhibition of this classof oncogenes, e.g., using antisense nucleotide sequences, iscontemplated for use in the present invention in aspects whereinenhancement of apoptosis is desired (U.S. Pat. Nos. 5,650,491;5,539,094; and 5,583,034; each incorporated herein by reference).

[0536] Other compositions that may be delivered by the antibodies of thepresent invention include genes encoding the tumor necrosis factorrelated apoptosis inducing ligand termed TRAIL, and the TRAILpolypeptide (U.S. Pat. No. 5,763,223; incorporated herein by reference);the 24 kD apoptosis-associated protease of U.S. Pat. No. 5,605,826(incorporated herein by reference); Fas-associated factor 1, FAF1 (U.S.Pat. No. 5,750,653; incorporated herein by reference). Also contemplatedfor use in these aspects of the present invention is the provision ofinterleukin-1β-converting enzyme and family members, which are alsoreported to stimulate apoptosis.

[0537] Compounds such as carbostyril derivatives (U.S. Pat. Nos.5,672,603; and 5,464,833; each incorporated herein by reference);branched apogenic peptides (U.S. Pat. No. 5,591,717; incorporated hereinby reference); phosphotyrosine inhibitors and non-hydrolyzablephosphotyrosine analogs (U.S. Pat. Nos. 5,565,491; and 5,693,627; eachincorporated herein by reference); agonists of RXR retinoid receptors(U.S. Pat. No. 5,399,586; incorporated herein by reference); and evenantioxidants (U.S. Pat. No. 5,571,523; incorporated herein by reference)may also be used. Tyrosine kinase inhibitors, such as genistein, mayalso be linked to the antibodies of the present invention (as supportedby U.S. Pat. No. 5,587,459; incorporated herein by reference).

[0538] F7. Anti-Viral Agents

[0539] As anionic phospholipids and aminophospholipids, particularly PSand PE, become exposed on virally infected cells, the antibodies of theinvention, such as the 9D2 and 3G4 (ATCC 4545) antibodies, may also belinked to any one or more anti-viral agents. Additional reasonsunderlying these aspects of the invention, and the advantages thereof,are described in more detail below in regard to the PE-binding peptide,anti-viral conjugates.

[0540] Exemplary anti-viral agents are for linking to antibodies orpeptides are also described in more detail in connection with thePE-binding peptide, anti-viral conjugates of the invention. Any one ormore anti-viral agents, including those in Table G, may be conjugated toan antibody of the invention, as described herein. Such anti-viralagents may also be used separately in the combination anti-viraltherapies of the invention.

[0541] G. Biologically Functional Equivalents

[0542] Equivalents, or even improvements, of antibodies and effectorscan now be made, generally using the materials provided above as astarting point. Modifications and changes may be made in the structureof such an antibody and still obtain a molecule having like or otherwisedesirable characteristics. For example, certain amino acids maysubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity. These considerationsalso apply to toxins, anti-angiogenic agents, apoptosis-inducing agents,coagulants and the like.

[0543] Since it is the interactive capacity and nature of a protein thatdefines that protein's biological functional activity, certain aminoacid sequence substitutions can be made in a protein sequence (or ofcourse, the underlying DNA sequence) and nevertheless obtain a proteinwith like (agonistic) properties. It is thus contemplated that variouschanges may be made in the sequence of the antibodies or therapeuticagents (or underlying DNA sequences) without appreciable loss of theirbiological utility or activity. Biological functional equivalents madefrom mutating an underlying DNA sequence can be made using the codoninformation provided herein in Table A, and the supporting technicaldetails on site-specific mutagenesis.

[0544] It also is well understood by the skilled artisan that, inherentin the definition of a “biologically functional equivalent” protein orpeptide, is the concept that there is a limit to the number of changesthat may be made within a defined portion of the molecule and stillresult in a molecule with an acceptable level of equivalent biologicalactivity. Biologically functional equivalent proteins and peptides arethus defined herein as those proteins and peptides in which certain, notmost or all, of the amino acids may be substituted. Of course, aplurality of distinct proteins/peptides with different substitutions mayeasily be made and used in accordance with the invention.

[0545] Amino acid substitutions are generally based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

[0546] In making more quantitative changes, the hydropathic index ofamino acids may be considered. Each amino acid has been assigned ahydropathic index on the basis of their hydrophobicity and chargecharacteristics, these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0547] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte and Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0548] It is thus understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent protein. As detailed in U.S. Pat. No. 4,554,101(incorporated herein by reference), the following hydrophilicity valueshave been assigned to amino acid residues: arginine (+3.0); lysine(+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

[0549] In making changes based upon hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within +0.5 are even more particularly preferred.

[0550] H. Conjugation

[0551] Antibodies to aminophospholipids and anionic phospholipids,including selected anti-PS antibodies with improved properties, such as9D2 and 3G4 (ATCC 4545), may be conjugated or attached to, oroperatively associated with, anti-cellular and cytotoxic agents toprepare “immunotoxins”; to coagulants, either directly or indirectly, toprepare “coaguligands”; or to anti-viral agents, such as nucleosides, toprepare anti-viral immunoconjugates or “immunovirocides”. PE-bindingpeptides such as duramycin may also be conjugated or attached to, oroperatively associated with, inert carriers, targeting agents oranti-viral agents, to prepare a range of PE-binding peptide derivativesand anti-viral peptide conjugates.

[0552] Although covalent linkages are preferred, other means ofoperative attachment may also be used. For example, linked constructsmay be generated using avidin:biotin bridges. In addition to theknowledge available to those of ordinary skill in the art, co-owned U.S.Pat. No. 6,093,399 is specifically incorporated herein by reference forpurposes of even further describing and enabling the use ofavidin:biotin in the operative attachment of antibodies and targetingagents to biological and therapeutic agents.

[0553] The two agents may also be joined by a second binding region,preferably an antibody or antigen binding region thereof. This isexemplified by coaguligands wherein the targeting agent is linked to thecoagulant via a second binding region (U.S. Pat. Nos. 6,093,399,6,004,555, 5,877,289, and 6,036,955, each specifically incorporatedherein by reference), which have been made and used successfully in thetreatment of cancer. Where the first targeting agent is an antibody orantigen binding region, the use of a second binding region that is alsoan antibody, or antigen binding region, results in a bispecific antibodyconstruct. The preparation and use of bispecific antibodies in generalis well known in the art, and is further disclosed herein.

[0554] Immunoconjugate technology is now generally known in the art.However, certain advantages may be achieved through the application ofcertain preferred technology, both in the preparation and purificationfor subsequent clinical administration. For example, while IgG basedconstructs will typically exhibit better binding capability and slowerblood clearance than their Fab′ counterparts, Fab′ fragment-basedconstructs will generally exhibit better tissue penetrating capability.

[0555] Additionally, while numerous types of disulfide-bond containinglinkers are known that can be successfully employed in antibody andpeptide conjugation, certain linkers will generally be preferred overother linkers, based on differing pharmacological characteristics andcapabilities. For example, linkers that contain a disulfide bond that issterically “hindered” are to be preferred, due to their greaterstability in vivo, thus preventing release of the coagulant prior tobinding at the site of action.

[0556] Each type of cross-linker, as well as how the cross-linking isperformed, will tend to vary the pharmacodynamics of the resultantconjugate. One may desire to have a conjugate that will remain intactunder conditions found everywhere in the body except the intended siteof action, at which point it is desirable that the conjugate have good“release” characteristics. Therefore, the particular cross-linkingscheme, including in particular the particular cross-linking reagentused and the structures that are cross-linked, will be of somesignificance.

[0557] Depending on the specific agents to be conjugated, it may benecessary or desirable to provide a peptide spacer operatively attachingthe antibody or PE-binding peptide and the second or therapeutic agent.Cetain peptide spacers are capable of folding into a disulfide-bondedloop structure. Proteolytic cleavage within the loop would then yield aheterodimeric polypeptide wherein the antibody and the therapeutic agentare linked by only a single disulfide bond. An example of such a toxinis a Ricin A-chain toxin.

[0558] When certain other toxin compounds are utilized, a non-cleavablepeptide spacer may be provided to operatively attach the antibody andthe toxin compound of the fusion protein. Toxins which may be used inconjunction with non-cleavable peptide spacers are those which may,themselves, be converted by proteolytic cleavage, into a cytotoxicdisulfide-bonded form. An example of such a toxin compound is aPseudonomas exotoxin compound.

[0559] A variety of chemotherapeutic and other pharmacological agentshave now been successfully conjugated to antibodies and shown tofunction pharmacologically. Exemplary antineoplastic agents that havebeen investigated include doxorubicin, daunomycin, methotrexate,vinblastine, and various others. Moreover, the attachment of otheragents such as neocarzinostatin, macromycin, trenimon and α-amanitin hasbeen described. These attachment methods can be adapted for useherewith.

[0560] Any covalent linkage to the antibody or PE-binding peptide shouldideally be made at a site distinct from the functional site(s). Thecompositions are thus “linked” in any operative manner that allows eachregion to perform its intended function without significant impairment,in particular, so that the resultant construct still binds to theintended antigen or to PE and so that the attached agent substantiallymaintains biological activity and/or recovers biological activity whenreleased from the construct.

[0561] Attachment of biological agents via the carbohydrate moieties onantibodies is also contemplated. Glycosylation, both O-linked andN-linked, naturally occurs in antibodies. Recombinant antibodies can bemodified to recreate or create additional glycosylation sites ifdesired, which is simply achieved by engineering the appropriate aminoacid sequences (such as Asn-X-Ser, Asn-X-Thr, Ser, or Thr) into theprimary sequence of the antibody.

[0562] H1. Biochemical Cross-Linkers

[0563] In additional to the general information provided above,antibodies or PE-binding peptides may be conjugated to therapeutic orother agents using certain preferred biochemical cross-linkers.Cross-linking reagents are used to form molecular bridges that tietogether functional groups of two different molecules. To link twodifferent proteins in a step-wise manner, hetero-bifunctionalcross-linkers can be used that eliminate unwanted homopolymer formation.Exemplary hetero-bifunctional cross-linkers are referenced in Table C.TABLE C HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Length LinkerReactive Toward Advantages and Applications after cross-linking SMPTPrimary amines Greater stability 11.2 A Sulfhydryls SPDP Primary aminesThiolation  6.8 A Sulfhydryls Cleavable cross-linking LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Sulfo-LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Water-soluble SMCC Primaryamines Stable maleimide reactive group 11.6 A SulfhydrylsEnzyme-antibody conjugation Hapten-carrier protein conjugationSulfo-SMCC Primary amines Stable maleimide reactive group 11.6 ASulfhydryls Water-soluble Enzyme-antibody conjugation MBS Primary aminesEnzyme-antibody conjugation  9.9 A Sulfhydryls Hapten-carrier proteinconjugation Sulfo-MBS Primary amines Water-soluble  9.9 A SulfhydrylsSIAB Primary amines Enzyme-antibody conjugation 10.6 A SulfhydrylsSulfo-SIAB Primary amines Water-soluble 10.6 A Sulfhydryls SMPB Primaryamines Extended spacer arm 14.5 A Sulfhydryls Enzyme-antibodyconjugation Sulfo-SMPB Primary amines Extended spacer arm 14.5 ASulfhydryls Water-soluble EDC/Sulfo-NHS Primary amines Hapten-Carrierconjugation  0 Carboxyl groups ABH Carbohydrates Reacts with sugargroups 11.9 A Nonselective

[0564] Hetero-bifunctional cross-linkers contain two reactive groups:one generally reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other generally reacting with a thiol group (e.g.,pyridyl disulfide, maleimides, halogens, etc.). Through the primaryamine reactive group, the cross-linker may react with the lysineresidue(s) of one protein (e.g., the selected antibody, fragment orPE-binding peptide) and through the thiol reactive group, thecross-linker, already tied up to the first protein, reacts with thecysteine residue (free sulfhydryl group) of the other protein.

[0565] Compositions therefore generally have, or are derivatized tohave, a functional group available for cross-linking purposes. Thisrequirement is not considered to be limiting in that a wide variety ofgroups can be used in this manner. For example, primary or secondaryamine groups, hydrazide or hydrazine groups, carboxyl alcohol,phosphate, carbamate, or alkylating groups may be used for binding orcross-linking.

[0566] The spacer arm between the two reactive groups of a cross-linkersmay have various length and chemical compositions. A longer spacer armallows a better flexibility of the conjugate components while someparticular components in the bridge (e.g., benzene group) may lend extrastability to the reactive group or an increased resistance of thechemical link to the action of various aspects (e.g., disulfide bondresistant to reducing agents). The use of peptide spacers, such asL-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.

[0567] It is preferred that a cross-linker having reasonable stabilityin blood will be employed. Numerous types of disulfide-bond containinglinkers are known that can be successfully employed in conjugation.Linkers that contain a disulfide bond that is sterically hindered mayprove to give greater stability in vivo, preventing release of the agentprior to binding at the site of action. These linkers are thus onepreferred group of linking agents.

[0568] One of the most preferred cross-linking reagents is SMPT, whichis a bifunctional cross-linker containing a disulfide bond that is“sterically hindered” by an adjacent benzene ring and methyl groups. Itis believed that steric hindrance of the disulfide bond serves afunction of protecting the bond from attack by thiolate anions such asglutathione which can be present in tissues and blood, and thereby helpin preventing decoupling of the conjugate prior to the delivery of theattached agent to the tumor site. It is contemplated that the SMPT agentmay also be used in connection with the conjugates of this invention.

[0569] The SMPT cross-linking reagent, as with many other knowncross-linking reagents, lends the ability to cross-link functionalgroups such as the SH of cysteine or primary amines (e.g., the epsilonamino group of lysine). Another possible type of cross-linker includesthe hetero-bifunctional photoreactive phenylazides containing acleavable disulfide bond such as sulfosuccinimidyl-2-(p-azidosalicylamido) ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidylgroup reacts with primary amino groups and the phenylazide (uponphotolysis) reacts non-selectively with any amino acid residue.

[0570] In addition to hindered cross-linkers, non-hindered linkers canalso be employed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane. The use of such cross-linkers is wellunderstood in the art.

[0571] Once conjugated, the conjugate is separated from unconjugatedantibodies or peptides and other agents and from other contaminants. Alarge a number of purification techniques are available for use inproviding conjugates of a sufficient degree of purity to render themclinically useful. Purification methods based upon size separation, suchas gel filtration, gel permeation or high performance liquidchromatography, will generally be of most use. Other chromatographictechniques, such as Blue-Sepharose separation, may also be used.

[0572] H2. Biologically Releasable Linkers

[0573] Although it is preferred that any linking moiety will havereasonable stability in blood, to prevent substantial release of theattached therapeutic agent before targeting to the disease, e.g., tumorsite, in certain aspects, the use of biologically-releasable bondsand/or selectively cleavable spacers or linkers is contemplated.“Biologically-releasable bonds” and “selectively cleavable spacers orlinkers” still have reasonable stability in the circulation.

[0574] The antibodies or PE-binding peptides in accordance with theinvention may thus be linked to one or more therapeutic or second agentsvia a biologically-releasable bond. Any form of targeting agent orantibody may be employed, including intact antibodies, although ScFvfragments will be preferred in certain embodiments.

[0575] “Biologically-releasable bonds” or “selectively hydrolyzablebonds” include all linkages that are releasable, cleavable orhydrolyzable only or preferentially under certain conditions. Thisincludes disulfide and trisulfide bonds and acid-labile bonds, asdescribed in U.S. Pat. Nos. 5,474,765 and 5,762,918, each specificallyincorporated herein by reference.

[0576] The use of an acid sensitive spacer for attachment of atherapeutic agent to an antibody or PE-binding peptide of the inventionis particularly contemplated. In such embodiments, the therapeuticagents are released within the acidic compartments inside a cell. It iscontemplated that acid-sensitive release may occur extracellularly, butstill after specific targeting, preferably to the tumor site or virallyinfected cell. Certain currently preferred examples include antibodieslinked to colchicine or doxorubicin via an acid sensitive spacer.Attachment via carbohydrate moieties of antibodies is also contemplated.In such embodiments, the therapeutic agent are released within theacidic compartments inside a cell.

[0577] The antibody or PE-binding peptide may also be derivatized tointroduce functional groups permitting the attachment of the therapeuticagents through a biologically releasable bond. The antibody orPE-binding peptide may thus be derivatized to introduce side chainsterminating in hydrazide, hydrazine, primary amine or secondary aminegroups. Therapeutic agents may be conjugated through a Schiffs baselinkage, a hydrazone or acyl hydrazone bond or a hydrazide linker (U.S.Pat. Nos. 5,474,765 and 5,762,918, each specifically incorporated hereinby reference).

[0578] Also as described in U.S. Pat. Nos. 5,474,765 and 5,762,918, eachspecifically incorporated herein by reference, the antibody orPE-binding peptide may be operatively attached to the therapeutic agentthrough one or more biologically releasable bonds that areenzyme-sensitive bonds, including peptide bonds, esters, amides,phosphodiesters and glycosides.

[0579] Certain preferred aspects of the invention concern the use ofpeptide linkers that include at least a first cleavage site for apeptidase and/or proteinase that is preferentially located within adisease site, particularly within the tumor environment. The antibody-or peptide-mediated delivery of the attached therapeutic agent thusresults in cleavage specifically within the disease site or tumorenvironment, resulting in the specific release of the active therapeuticagent. Certain peptide linkers will include a cleavage site that isrecognized by one or more enzymes involved in remodeling.

[0580] Peptide linkers that include a cleavage site for urokinase,pro-urokinase, plasmin, plasminogen, TGFβ, staphylokinase, Thrombin,Factor IXa, Factor Xa or a metalloproteinase, such as an interstitialcollagenase, a gelatinase or a stromelysin, are particularly preferred.U.S. Pat. Nos. 6,004,555, 5,877,289, and 6,093,399 are specificallyincorporated herein by reference for the purpose of further describingand enabling how to make and use immunoconjugates comprisingbiologically-releasable bonds and selectively-cleavable linkers andpeptides. U.S. Pat. No. 5,877,289 is particularly incorporated herein byreference for the purpose of further describing and enabling how to makeand use immunoconjugates that comprise a selectively-cleavable peptidelinker that is cleaved by urokinase, plasmin, Thrombin, Factor IXa,Factor Xa or a metalloproteinase, such as an interstitial collagenase, agelatinase or a stromelysin, within a tumor environment.

[0581] Currently preferred selectively-cleavable peptide linkers arethose that include a cleavage site for plasmin or a metalloproteinase(also known as “matrix metalloproteases” or “MMPs”), such as aninterstitial collagenase, a gelatinase or a stromelysin. Additionalpeptide linkers that may be advantageously used in connection with thepresent invention include, for example, plasmin cleavable sequences,such as those cleavable by pro-urokinase, TGFβ, plasminogen andstaphylokinase; Factor Xa cleavable sequences; MMP cleavable sequences,such as those cleavable by gelatinase A; collagenase cleavablesequences, such as those cleavable by calf skin collagen (α1(I) chain),calf skin collagen (α2(I) chain), bovine cartilage collagen (α1(II)chain), human liver collagen (α1(III) chain), human α₂M, human PZP, ratα₁M, rat α₂M, rat α₁I₃(2J), rat α₁I₃(27J), and the human fibroblastcollagenase autolytic cleavage sites. In addition to the knowledgeavailable to those of ordinary skill in the art, the text and sequencesfrom Table B2 in co-owned U.S. Pat. Nos. 6,342,219, 6,524,583, 6,342,221and 6,416,758, are specifically incorporated herein by reference forpurposes of even further describing and enabling the use of suchcleavable sequences.

[0582] H3. Bispecific Antibodies

[0583] Bispecific antibodies in general may be employed, so long as onearm binds to an aminophospholipid or anionic phospholipid and thebispecific antibody is attached, at a site distinct from the antigenbinding site, to a therapeutic agent.

[0584] In general, the preparation of bispecific antibodies is also wellknown in the art. One method involves the separate preparation ofantibodies having specificity for the aminophospholipid or anionicphospholipid, on the one hand, and a therapeutic agent on the other.Peptic F(ab′γ)₂ fragments are prepared from the two chosen antibodies,followed by reduction of each to provide separate Fab′γsH fragments. TheSH groups on one of the two partners to be coupled are then alkylatedwith a cross-linking reagent such as O-phenylenedimaleimide to providefree maleimide groups on one partner. This partner may then beconjugated to the other by means of a thioether linkage, to give thedesired F(ab′γ)₂ heteroconjugate. Other techniques are known whereincross-linking with SPDP or protein A is carried out, or a trispecificconstruct is prepared.

[0585] Another method for producing bispecific antibodies is by thefusion of two hybridomas to form a quadroma. As used herein, the term“quadroma” is used to describe the productive fusion of two B cellhybridomas. Using now standard techniques, two antibody producinghybridomas are fused to give daughter cells, and those cells that havemaintained the expression of both sets of clonotype immunoglobulin genesare then selected.

[0586] A preferred method of generating a quadroma involves theselection of an enzyme deficient mutant of at least one of the parentalhybridomas. This first mutant hybridoma cell line is then fused to cellsof a second hybridoma that had been lethally exposed, e.g., toiodoacetamide, precluding its continued survival. Cell fusion allows forthe rescue of the first hybridoma by acquiring the gene for its enzymedeficiency from the lethally treated hybridoma, and the rescue of thesecond hybridoma through fusion to the first hybridoma Preferred, butnot required, is the fusion of immunoglobulins of the same isotype, butof a different subclass. A mixed subclass antibody permits the use if analternative assay for the isolation of a preferred quadroma.

[0587] In more detail, one method of quadroma development and screeninginvolves obtaining a hybridoma line that secretes the first chosen MAband making this deficient for the essential metabolic enzyme,hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To obtaindeficient mutants of the hybridoma, cells are grown in the presence ofincreasing concentrations of 8-azaguanine (1×10⁻⁷M to 1×10⁻⁵M). Themutants are subcloned by limiting dilution and tested for theirhypoxanthine/aminopterin/thymidine (HAT) sensitivity. The culture mediummay consist of, for example, DMEM supplemented with 10% FCS, 2 mML-Glutamine and 1 mM penicillin-streptomycin.

[0588] A complementary hybridoma cell line that produces the seconddesired MAb is used to generate the quadromas by standard cell fusiontechniques. Briefly, 4.5×10⁷ HAT-sensitive first cells are mixed with2.8×10⁷ HAT-resistant second cells that have been pre-treated with alethal dose of the irreversible biochemical inhibitor iodoacetamide (5mM in phosphate buffered saline) for 30 minutes on ice before fusion.Cell fusion is induced using polyethylene glycol (PEG) and the cells areplated out in 96 well microculture plates. Quadromas are selected usingHAT-containing medium. Bispecific antibody-containing cultures areidentified using, for example, a solid phase isotype-specific ELISA andisotype-specific immunofluorescence staining.

[0589] In one identification embodiment to identify the bispecificantibody, the wells of microtiter plates (Falcon, Becton DickinsonLabware) are coated with a reagent that specifically interacts with oneof the parent hybridoma antibodies and that lacks cross-reactivity withboth antibodies. The plates are washed, blocked, and the supernatants(SNs) to be tested are added to each well. Plates are incubated at roomtemperature for 2 hours, the supernatants discarded, the plates washed,and diluted alkaline phosphatase-anti-antibody conjugate added for 2hours at room temperature. The plates are washed and a phosphatasesubstrate, e.g., P-Nitrophenyl phosphate (Sigma, St. Louis) is added toeach well. Plates are incubated, 3N NaOH is added to each well to stopthe reaction, and the OD₄₁₀ values determined using an ELISA reader.

[0590] In another identification embodiment, microtiter platespre-treated with poly-L-lysine are used to bind one of the target cellsto each well, the cells are then fixed, e.g. using 1% glutaraldehyde,and the bispecific antibodies are tested for their ability to bind tothe intact cell. In addition, FACS, immunofluorescence staining,idiotype specific antibodies, antigen binding competition assays, andother methods common in the art of antibody characterization may be usedin conjunction with the present invention to identify preferredquadromas.

[0591] Following the isolation of the quadroma, the bispecificantibodies are purified away from other cell products. This may beaccomplished by a variety of protein isolation procedures, known tothose skilled in the art of immunoglobulin purification. Means forpreparing and characterizing antibodies are well known in the art (See,e.g., Antibodies: A Laboratory Manual, 1988).

[0592] For example, supernatants from selected quadromas are passed overprotein A or protein G sepharose columns to bind IgG (depending on theisotype). The bound antibodies are then eluted with, e.g. a pH 5.0citrate buffer. The elute fractions containing the BsAbs, are dialyzedagainst an isotonic buffer. Alternatively, the eluate is also passedover an anti-immunoglobulin-sepharose column. The BsAb is then elutedwith 3.5 M magnesium chloride. BsAbs purified in this way are thentested for binding activity by, e.g., an isotype-specific ELISA andimmunofluorescence staining assay of the target cells, as describedabove.

[0593] Purified BsAbs and parental antibodies may also be characterizedand isolated by SDS-PAGE electrophoresis, followed by staining withsilver or Coomassie. This is possible when one of the parentalantibodies has a higher molecular weight than the other, wherein theband of the BsAbs migrates midway between that of the two parentalantibodies. Reduction of the samples verifies the presence of heavychains with two different apparent molecular weights.

[0594] H4. Fusion Proteins and Recombinant Expression

[0595] Antibodies to aminophospholipids and anionic phospholipids,including the 9D2 and 3G4 (ATCC 4545) antibodies and other competingantibodies with improved properties, and PE-binding peptides, can alsobe used to create fusion proteins using molecular biological techniques.Any fusion protein may be designed and made using any of the antibodies,PE-binding peptides and second or therapeutic agents disclosed hereinand those known in the art. The fusion protein technology is readilyadaptable to prepare fusion proteins with other modifications, such asoptimizations in CDR sequences, linkage via a selectively cleavablepeptide sequence, and such like.

[0596] The use of recombinant DNA techniques to achieve such ends is nowstandard practice to those of skill in the art. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. DNA and RNA synthesismay, additionally, be performed using an automated synthesizers (see,for example, the techniques described in Sambrook et al., 1989).

[0597] The preparation of such a fusion protein generally entails thepreparation of a first and second DNA coding region and the functionalligation or joining of such regions, in frame, to prepare a singlecoding region that encodes the desired fusion protein. In the presentcontext, the antibody sequence will be joined in frame with a DNAsequence encoding a therapeutic agent. It is not generally believed tobe particularly relevant which portion of the immunoconjugate isprepared as the N-terminal region or as the C-terminal region.

[0598] Once the desired coding region has been produced, an expressionvector is created. Expression vectors contain one or more promotersupstream of the inserted DNA regions that act to promote transcriptionof the DNA and to thus promote expression of the encoded recombinantprotein. This is the meaning of “recombinant expression”.

[0599] To obtain a so-called “recombinant” version of theimmunoconjugate, the vector is expressed in a recombinant cell. Theengineering of DNA segment(s) for expression in a prokaryotic oreukaryotic system may be performed by techniques generally known tothose of skill in recombinant expression. It is believed that virtuallyany expression system may be employed in expression.

[0600] The immunoconjugates of the invention may be successfullyexpressed in eukaryotic expression systems, e.g., CHO cells, however, itis envisioned that bacterial expression systems, such as E. coli pQE-60will be particularly useful for the large-scale preparation andsubsequent purification of the constructs. cDNAs may also be expressedin bacterial systems, with the encoded proteins being expressed asfusions with β-galactosidase, ubiquitin, Schistosoma japonicumglutathione S-transferase, and the like. It is believed that bacterialexpression will have advantages over eukaryotic expression in terms ofease of use and quantity of materials obtained thereby.

[0601] In terms of microbial expression, U.S. Pat. Nos. 5,583,013;5,221,619; 4,785,420; 4,704,362; and 4,366,246 are incorporated hereinby reference for the purposes of even further supplementing the presentdisclosure in connection with the expression of genes in recombinanthost cells.

[0602] Recombinantly produced immunoconjugates may be purified andformulated for human administration. Alternatively, nucleic acidsencoding the immunoconjugates may be delivered via gene therapy.Although naked recombinant DNA or plasmids may be employed, the use ofliposomes or vectors is preferred. The ability of certain viruses toenter cells via receptor-mediated endocytosis and to integrate into thehost cell genome and express viral genes stably and efficiently havemade them attractive candidates for the transfer of foreign genes intomammalian cells. Preferred gene therapy vectors for use in the presentinvention will generally be viral vectors.

[0603] Retroviruses have promise as gene delivery vectors due to theirability to integrate their genes into the host genome, transferring alarge amount of foreign genetic material, infecting a broad spectrum ofspecies and cell types and of being packaged in special cell-lines.Other viruses, such as adenovirus, herpes simplex viruses (HSV),cytomegalovirus (CMV), and adeno-associated virus (AAV), such as thosedescribed by U.S. Pat. No. 5,139,941 (incorporated herein by reference),may also be engineered to serve as vectors for gene transfer.

[0604] Although some viruses that can accept foreign genetic materialare limited in the number of nucleotides they can accommodate and in therange of cells they infect, these viruses have been demonstrated tosuccessfully effect gene expression. However, adenoviruses do notintegrate their genetic material into the host genome and therefore donot require host replication for gene expression, making them ideallysuited for rapid, efficient, heterologous gene expression. Techniquesfor preparing replication-defective infective viruses are well known inthe art.

[0605] In certain further embodiments, the gene therapy vector will beHSV. A factor that makes HSV an attractive vector is the size andorganization of the genome. Because HSV is large, incorporation ofmultiple genes or expression cassettes is less problematic than in othersmaller viral systems. In addition, the availability of different viralcontrol sequences with varying performance (e.g., temporal, strength)makes it possible to control expression to a greater extent than inother systems. It also is an advantage that the virus has relatively fewspliced messages, further easing genetic manipulations. HSV also isrelatively easy to manipulate and can be grown to high titers.

[0606] Of course, in using viral delivery systems, one will desire topurify the virion sufficiently to render it essentially free ofundesirable contaminants, such as defective interfering viral particlesor pyrogens such that it will not cause any untoward reactions in thecell, animal or individual receiving the vector construct. A preferredmeans of purifying the vector involves the use of buoyant densitygradients, such as cesium chloride gradient centrifugation.

[0607] I. Binding, Functional and Screening Assays

[0608] Although the present invention has significant utility in animaland human treatment regimens, it also has many other specific andcredible uses, including practical uses in many in vitro embodiments.Certain of these uses are related to the specific binding properties ofthe antibodies, peptides and immunoconjugates. In that each of theconstructs of the invention include at least one antibody or peptidecomponent that binds to an aminophospholipid and/or an anionicphospholipid, they may be used in a variety of binding embodiments,including useful binding assays.

[0609] The presence of an attached agent, where relevant, althoughproviding advantageous properties, does not negate the utility of thefirst antibody or peptide regions in any binding assay. Suitably usefulbinding assays thus include those commonly employed in the art, such asin immunoblots, Western blots, dot blots, RIAs, ELISAs,immunohistochemistry, fluorescent activated cell sorting (FACS),immunoprecipitation, affinity chromatography, and the like, as furtherdescribed herein.

[0610] Certain standard binding assays are those in which an antigen isimmobilized onto a solid support matrix, e.g., nitrocellulose, nylon ora combination thereof, such as in immunoblots, Western blots, ELISAs andrelated assays. Other important assays are those using cells, whereinthe components of the present invention can be used to assay for cellswith aminophospholipids and/or anionic phospholipids at the cellsurface. Such assays can be applied in pre-clinical testing, e.g.,regarding the design of drugs, testing the mechanism of action and/orselecting therapeutic agents for combined use.

[0611] Further in vitro assays are useful in the diagnosis of diseasesconnected with aberrant cell activation and/or apoptosis, whereintesting for the presence of aminophospholipids and/or anionicphospholipids at the cell surface would be particularly useful. Theconstructs of the invention may thus be used in conjunction with bothfresh-frozen and formalin-fixed, paraffin-embedded tissue blocks inimmunohistochemistry; in fluorescent activated cell sorting, flowcytometry or flow microfluorometry.

[0612] They constructs of the invention have further practical uses inimmunoprecipitation, antigen purification embodiments, such as affinitychromatography, even including, in cases of bispecific antibodies, theone-step rapid purification of one or more antigens at the same time;and in many other binding assays that will be known to those of skill inthe art given the information presented herein.

[0613] Yet further practical uses of the present constructs are ascontrols in functional assays, including many in vitro and ex vivoassays and systems. As the binding and functional properties of theantibodies, peptides and conjugates of the invention are particularlyspecific, as disclosed herein, such “control” uses are actuallyextremely valuable. The assays that benefit from such a practicalapplication of the present invention include, for example, assaysconcerning detection of aminophospholipids and/or anionic phospholipidsat the cell surface.

[0614] These assays systems can also be developed into in vitro or exvivo drug screening assays, wherein the present provision of biologicalmaterials with well defined properties is particularly important. Forexample, in using the constructs of the present invention as positivecontrols in the selection of small molecules that have similar,equivalent or improved binding properties, e.g., in drug screening anddevelopment.

[0615] The binding assays and systems of the invention can also bedeveloped into in vitro or ex vivo drug screening assays, wherein thepresent provision of biological materials with well defined properties,as in the antibodies disclosed herein, is particularly important. Forexample, in using the constructs of the present invention as positivecontrols in the selection of small molecules that have similar,equivalent or improved binding properties, e.g., in drug screening anddevelopment.

[0616] In this regard, the invention further provides methods ofscreening for compounds that mimic the binding and activity of theantibodies disclosed herein, preferably the 9D2 or 3G4 antibodies, andmost preferably the 3G4 (ATCC 4545) antibody. As the antibodies of theinvention bind to aminophospholipids and anionic phospholipids,preferably PS and PE, preferred screening methods are those that testcompounds for the ability to inhibit the binding of the antibodies toone or more aminophospholipids or anionic phospholipids, such as PS andPE. The methods are suitable for use in screening for low molecularweight compounds for use as drugs that mimic the anti-tumor,anti-vascular and/or anti-viral activities of the antibodies.

[0617] The “screening assays or methods” of the invention are conductedon the same principles as the techniques employed in testing competingor cross-reactive antibodies, as taught herein and further exemplifiedin the working examples. The starting materials, steps, qualitative andquantitative guidelines for use in the antibody competition assays maythus be readily adapted for use in the present screening assays,particularly in light of the following information.

[0618] In the screening methods, the agents to be tested may be termed“candidate substances”. Candidate substances for screening in suchassays include those isolated from natural sources, including bacteria,fungi, plant sources, including leaves and bark, soil and marinesamples. Other candidate substances that may be screened in this mannerare those derived from chemical compositions or man-made compounds,particularly those in large chemical libraries.

[0619] As with the methods for identifying competing antibodies, thescreening assays test the ability of a candidate substance to inhibitbinding of a “control positive antibody”, such as the 3G4 antibody, toan aminophospholipid or anionic phospholipid, preferably PS. Antibodybinding is first measured in the absence of the candidate substance,which is preferably repeated once in each assay, but can also bereferred to from a known standard. The candidate substance(s) are thenadmixed with samples of the antibody and the ability of the antibody tobind to the target, preferably PS, is determined in the presence of thecandidate substance. A substance that reduces binding, and preferablysignificantly reduces binding, in comparison to the level in the absenceof the substance, is indicative of a candidate substance withcompetitive capability. Such substances are “positive candidatesubstances” and are continued for further development.

[0620] In preferred embodiments, the screening is conducted usingPS-coated microtiter plates. A high throughput screening procedure isconsidered most useful, many suitable examples of which are known andmay now be used, in light of the motivation in the present disclosure,and in conjunction with the reagents provided by the invention. Oneexample of high throughput screening concerns testing compounds for theability to inhibit the binding of luciferase-labeled antibodies, such asluciferase-labeled 3G4, to PS-coated microtiter plates.Luciferase-labeled 3G4 antibodies, and kits comprising such antibodies,are thus further components within the overall invention.

[0621] It is expected that suitable positive candidate substances willbe identified using the screening methods of the invention(notwithstanding the fact that the screening assays are useful inthemselves, even without identifying effective candidate substances). Ifdesired, chemical or biological modifications can be made to thepositive candidate substances first identified, and the modifiedversions or “analogs” re-screened to continue the process of selectingthe most useful agents, e.g., to select more optimal inhibitorycompounds from chemical derivatives. Inhibitory activity can also beconfirmed by re-screening after modifications based upon mechanisticconsiderations. For example, as cross-linking PS or PE on the targetcells may be required for optimal biological activity in vivo, positivecandidate substances from the first screening assays may be linked toform dimers, trimers, oligomers or multimers and re-screened to confirminhibitory activity, preferably followed by further tests to confirmcross-linking in vitro.

[0622] J. Pharmaceutical Compositions

[0623] The therapeutic agents of the present invention will generally beformulated as pharmaceutical compositions. The pharmaceuticalcompositions will comprise a biologically or therapeutically effectiveamount of at least a first therapeutic agent of the invention, dissolvedor dispersed in a pharmaceutically acceptable carrier or aqueous medium.Combined therapeutics are also contemplated, and the same type ofunderlying pharmaceutical compositions may be employed for both singleand combined medicaments.

[0624] The phrases “pharmaceutically or pharmacologically acceptable”refer to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, or a human, as appropriate. Veterinary uses are equally includedwithin the invention and “pharmaceutically acceptable” formulationsinclude formulations for both clinical and/or veterinary use.

[0625] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. For human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards. Supplementary activeingredients can also be incorporated into the compositions.

[0626] “Unit dosage” formulations are those containing a dose orsub-dose of the administered ingredient adapted for a particular timeddelivery. For example, exemplary “unit dosage” formulations are thosecontaining a daily dose or unit or daily sub-dose or a weekly dose orunit or weekly sub-dose and the like.

[0627] J1. Injectable Formulations

[0628] The therapeutic agents of the invention will often be formulatedfor parenteral administration, particularly for tumor treatment, e.g.,formulated for injection via the intravenous, intramuscular,sub-cutaneous, transdermal, or other such routes, including peristalticadministration and direct instillation into a tumor or disease site(intracavity administration). The preparation of an aqueous compositionthat contains an antibody, immunoconjugate or peptide conjugate as anactive ingredient will be known to those of skill in the art in light ofthe present disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for using to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and thepreparations can also be emulsified.

[0629] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions; formulations including sesameoil, peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and fluid to theextent that syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.

[0630] The therapeutic agents can be formulated into a sterile aqueouscomposition in a neutral or salt form. Solutions of therapeutic agentsas free base or pharmacologically acceptable salts can be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein), and those that areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, trifluoroacetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like.

[0631] Suitable carriers include solvents and dispersion mediacontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants.

[0632] Under ordinary conditions of storage and use, all suchpreparations should contain a preservative to prevent the growth ofmicroorganisms. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

[0633] Prior to or upon formulation, the therapeutic agents should beextensively dialyzed to remove undesired small molecular weightmolecules, and/or lyophilized for more ready formulation into a desiredvehicle, where appropriate. Sterile injectable solutions are prepared byincorporating the active agents in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as desired, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle that contains the basic dispersionmedium and the required other ingredients from those enumerated above.

[0634] In the case of sterile powders for the preparation of sterileinjectable solutions, the preferred methods of preparation arevacuum-drying and freeze-drying techniques that yield a powder of theactive ingredient, plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0635] Suitable pharmaceutical compositions in accordance with theinvention will generally include an amount of the therapeutic agentadmixed with an acceptable pharmaceutical diluent or excipient, such asa sterile aqueous solution, to give a range of final concentrations,depending on the intended use. The techniques of preparation aregenerally well known in the art as exemplified by Remington'sPharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980,incorporated herein by reference. For human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biological Standards. Upon formulation, thetherapeutic agents will be administered in a manner compatible with thedosage formulation and in such amount as is therapeutically effective.

[0636] J2. Sustained Release Formulations

[0637] Formulations are easily administered in a variety of dosageforms, such as the type of injectable solutions described above, butother pharmaceutically acceptable forms are also contemplated, e.g.,tablets, pills, capsules or other solids for oral administration,suppositories, pessaries, nasal solutions or sprays, aerosols,inhalants, topical formulations, liposomal forms and the like. The typeof form for administration will be matched to the disease or disorder tobe treated.

[0638] Pharmaceutical “slow release” capsules or “sustained release”compositions or preparations may also be used. Slow release formulationsare generally designed to give a constant drug level over an extendedperiod and may be used to deliver therapeutic agents in accordance withthe present invention. The slow release formulations are typicallyimplanted in the vicinity of the disease site, for example, at the siteof a tumor or viral infection.

[0639] Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containingtherapeutic agents, which matrices are in the form of shaped articles,e.g., films or microcapsule. Examples of sustained-release matricesinclude polyesters; hydrogels, for example,poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol); polylactides,e.g., U.S. Pat. No. 3,773,919; copolymers of L-glutamic acid and γethyl-L-glutamate; non-degradable ethylene-vinyl acetate; degradablelactic acid-glycolic acid copolymers, such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate); and poly-D-(−)-3-hydroxybutyric acid.

[0640] While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated antibodies remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.,thus reducing biological activity and/or changing immunogenicity.Rational strategies are available for stabilization depending on themechanism involved. For example, if the aggregation mechanism involvesintermolecular S—S bond formation through thio-disulfide interchange,stabilization is achieved by modifying sulfhydryl residues, lyophilizingfrom acidic solutions, controlling moisture content, using appropriateadditives, developing specific polymer matrix compositions, and thelike.

[0641] J3. Liposomes and Nanocapsules

[0642] In certain embodiments, liposomes and/or nanoparticles may alsobe employed with the therapeutic agents. The formation and use ofliposomes is generally known to those of skill in the art, as summarizedbelow. The present invention provides particular combinations ofantibodies, liposomes and chemotherapeutic agents, which are describedbelow. In addition, a liposomal formulation may be used as a routinecomponent of any of the therapeutic agents of the overall invention.

[0643] Liposomes are formed from phospholipids that are dispersed in anaqueous medium and spontaneously form multilamellar concentric bilayervesicles (also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

[0644] Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

[0645] Liposomes interact with cells via four different mechanisms:Endocytosis by phagocytic cells of the reticuloendothelial system suchas macrophages and neutrophils; adsorption to the cell surface, eitherby nonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. Varying the liposome formulation can alter whichmechanism is operative, although more than one may operate at the sametime.

[0646] Nanocapsules can generally entrap compounds in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

[0647] J4. Ophthalmic Formulations

[0648] Many diseases of the eye, particularly those having an angiogeniccomponent, can be treated by the present invention. For example ocularneovascular disease, age-related macular degeneration, diabeticretinopathy, retinopathy of prematurity, corneal graft rejection,neovascular glaucoma, retrolental fibroplasias and other diseasesassociated with corneal neovascularization or retinal/choroidalneovascularization, as described hereinbelow.

[0649] The therapeutic agents of the present invention may thus beadvantageously employed in the preparation of pharmaceuticalcompositions suitable for use as ophthalmic solutions, including thosefor intravitreal and/or intracameral administration. For the treatmentof any of the foregoing or other disorders the therapeutic agents areadministered to the eye or eyes of the subject in need of treatment inthe form of an ophthalmic preparation prepared in accordance withconventional pharmaceutical practice, see for example “Remington'sPharmaceutical Sciences” 15th Edition, pages 1488 to 1501 (MackPublishing Co., Easton, Pa.).

[0650] The ophthalmic preparations will contain a therapeutic agent in aconcentration from about 0.01 to about 1% by weight, preferably fromabout 0.05 to about 0.5% in a pharmaceutically acceptable solution,suspension or ointment. Some variation in concentration will necessarilyoccur, depending on the particular compound employed, the condition ofthe subject to be treated and the like, and the person responsible fortreatment will determine the most suitable concentration for theindividual subject. The ophthalmic preparation will preferably be in theform of a sterile aqueous solution containing, if desired, additionalingredients, for example preservatives, buffers, tonicity agents,antioxidants and stabilizers, nonionic wetting or clarifying agents,viscosity-increasing agents and the like.

[0651] Suitable preservatives for use in such a solution includebenzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosaland the like. Suitable buffers include boric acid, sodium and potassiumbicarbonate, sodium and potassium borates, sodium and potassiumcarbonate, sodium acetate, sodium biphosphate and the like, in amountssufficient to maintain the pH at between about pH 6 and pH 8, andpreferably, between about pH 7 and pH 7.5. Suitable tonicity agents aredextran 40, dextran 70, dextrose, glycerin, potassium chloride,propylene glycol, sodium chloride, and the like, such that the sodiumchloride equivalent of the ophthalmic solution is in the range 0.9 plusor minus 0.2%.

[0652] Suitable antioxidants and stabilizers include sodium bisulfite,sodium metabisulfite, sodium thiosulfite, thiourea and the like.Suitable wetting and clarifying agents include polysorbate 80,polysorbate 20, poloxamer 282 and tyloxapol. Suitableviscosity-increasing agents include dextran 40, dextran 70, gelatin,glycerin, hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin,methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose and the like. Theophthalmic preparation will be administered topically to the eye of thesubject in need of treatment by conventional methods, for example in theform of drops or by bathing the eye in the ophthalmic solution.

[0653] J5. Topical Formulations

[0654] In the broadest sense, formulations for topical administrationinclude those for delivery via the mouth (buccal) and through the skin.“Topical delivery systems” also include transdermal patches containingthe ingredient to be administered. Delivery through the skin can furtherbe achieved by iontophoresis or electrotransport, if desired.

[0655] Formulations suitable for topical administration in the mouthinclude lozenges comprising the ingredients in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the ingredient to be administeredin a suitable liquid carrier.

[0656] Formulations suitable for topical administration to the skininclude ointments, creams, gels and pastes comprising the ingredient tobe administered in a pharmaceutical acceptable carrier. The formulationof therapeutic agents for topical use, such as in creams, ointments andgels, includes the preparation of oleaginous or water-soluble ointmentbases, will be well known to those in the art in light of the presentdisclosure. For example, these compositions may include vegetable oils,animal fats, and more preferably, semisolid hydrocarbons obtained frompetroleum. Particular components used may include white ointment, yellowointment, cetyl esters wax, oleic acid, olive oil, paraffin, petrolatum,white petrolatum, spermaceti, starch glycerite, white wax, yellow wax,lanolin, anhydrous lanolin and glyceryl monostearate. Variouswater-soluble ointment bases may also be used, including glycol ethersand derivatives, polyethylene glycols, polyoxyl 40 stearate andpolysorbates.

[0657] Formulations for rectal administration may be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate. Formulations suitable for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing in addition to the active ingredient suchcarriers as are known in the art to be appropriate.

[0658] J6. Nasal Formulations

[0659] Local delivery via the nasal and respiratory routes iscontemplated for treating various conditions, particularly for use inthe anti-viral treatment methods of the present invention. Thesedelivery routes are also suitable for delivering agents into thesystemic circulation. Formulations of active ingredients in carrierssuitable for nasal administration are therefore also included within theinvention, for example, nasal solutions, sprays, aerosols and inhalants.Where the carrier is a solid, the formulations include a coarse powderhaving a particle size, for example, in the range of 20 to 500 microns,which is administered, e.g., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose.

[0660] Suitable formulations wherein the carrier is a liquid are usefulin nasal administration. Nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays andare prepared so that they are similar in many respects to nasalsecretions, so that normal ciliary action is maintained. Thus, theaqueous nasal solutions usually are isotonic and slightly buffered tomaintain a pH of 5.5 to 6.5. In addition, antimicrobial preservatives,similar to those used in ophthalmic preparations, and appropriate drugstabilizers, if required, may be included in the formulation. Variouscommercial nasal preparations are known and include, for example,antibiotics and antihistamines and are used for asthma prophylaxis.

[0661] Inhalations and inhalants are pharmaceutical preparationsdesigned for delivering a drug or compound into the respiratory tree ofa patient. A vapor or mist is administered and reaches the affectedarea. This route can also be employed to deliver agents into thesystemic circulation. Inhalations may be administered by the nasal ororal respiratory routes. The administration of inhalation solutions isonly effective if the droplets are sufficiently fine and uniform in sizeso that the mist reaches the bronchioles.

[0662] Another group of products, also known as inhalations, andsometimes called insufflations, comprises finely powdered or liquiddrugs that are carried into the respiratory passages by the use ofspecial delivery systems, such as pharmaceutical aerosols, that hold asolution or suspension of the drug in a liquefied gas propellant. Whenreleased through a suitable valve and oral adapter, a metered does ofthe inhalation is propelled into the respiratory tract of the patient.Particle size is of major importance in the administration of this typeof preparation. It has been reported that the optimum particle size forpenetration into the pulmonary cavity is of the order of 0.5 to 7 μm.Fine mists are produced by pressurized aerosols and hence their use inconsidered advantageous.

[0663] K. Diagnostic and Therapeutic Kits

[0664] This invention also provides diagnostic and therapeutic kitscomprising at least a first therapeutic agent of the present invention,i.e., an antibody, immunoconjugate or peptide conjugate that binds to anaminophospholipid or anionic phospholipid, for use in treatment methods,combined treatment methods and/or in imaging and treatment embodiments.Such kits will generally contain, in at least a first suitable container(or container means), a pharmaceutically acceptable formulation of atleast one therapeutic agent, antibody, immunoconjugate or peptideconjugate that binds to an aminophospholipid or anionic phospholipid.The kits may include written or electronic instructions for use, e.g. inpre-clinical, clinical and/or veterinary embodiments.

[0665] The kits may also contain other compositions, pharmaceuticallyacceptable formulations and second biological and therapeutic agents,including those for combined therapy and/or for diagnostic and imaging.For example, such kits may contain any one or more of a range ofchemotherapeutic, radiotherapeutic or anti-angiogenic agents, anti-tumorcell, anti-tumor vasculature or anti-tumor stroma antibodies,immunotoxins or coaguligands, anti-viral agents and/or diagnosticcomponents or agents. Written or electronic instructions for use incombined therapy and/or for diagnosis and imaging may also be included.

[0666] The kits may have a single container that contains the firstantibody, immunoconjugate or peptide conjugate that binds to anaminophospholipid or anionic phospholipid, with or without anyadditional components, or they may have distinct containers for eachdesired agent. Where combined therapeutics are provided, a singlesolution may be pre-mixed, either in a molar equivalent combination, orwith one component in excess of the other. Alternatively, the primarytherapeutic agent of the invention and the second biological ortherapeutic agent, such as a second anti-cancer or anti-viral agent, kitmay be maintained separately within distinct containers of the kit priorto administration to a patient.

[0667] Diagnostic components will most often be maintained in at least asecond container, distinct from the other or first container thatcomprises the one or more therapeutic agents. The diagnostic kits mayinclude labeled antibodies or peptides that bind to the sameaminophospholipid or anionic phospholipid as the primary therapeuticagent, or any other agent suitable for diagnosing the disease to betreated. The kits may include diagnostic agents for use in vitro, foruse in vivo, or both such agent. The kits may include written orelectronic instructions for use, e.g. in pre-clinical, clinical and/orveterinary diagnostic embodiments.

[0668] For immunodetection in vitro, the antibodies may be bound to asolid support, such as a well of a microtitre plate, although antibodysolutions or powders for reconstitution are preferred. Theimmunodetection kits preferably comprise at least a firstimmunodetection reagent. The immunodetection reagents of the kit maytake any one of a variety of forms, including those detectable labelsthat are associated with or linked to the given antibody, such as usedin vivo. Detectable labels that are associated with or attached to asecondary binding ligand are also contemplated. Exemplary secondaryligands are those secondary antibodies that have binding affinity forthe first antibody.

[0669] Further suitable immunodetection reagents for use in the presentkits include the two-component reagent that comprises a secondaryantibody that has binding affinity for the first antibody, along with athird antibody that has binding affinity for the second antibody, thethird antibody being linked to a detectable label. A number of exemplarylabels are known in the art and all such labels may be employed inconnection with the present invention. These kits may containantibody-label conjugates either in fully conjugated form, in the formof intermediates, or as separate moieties to be conjugated by the userof the kit. The imaging kits will preferably comprise a targeting agentor antibody that is already attached to an in vivo detectable label.However, the label and attachment means could be separately supplied.

[0670] Either form of diagnostic kit may further comprise controlagents, such as suitably aliquoted biological compositions, whetherlabeled or unlabeled, as may be used to prepare a standard curve for adetection assay. The components of the kits may be packaged either inaqueous media or in lyophilized form.

[0671] When the components of the kit are provided in one or more liquidsolutions, the liquid solution is preferably an aqueous solution, with asterile aqueous solution being particularly preferred. However, thecomponents of the kit may be provided as dried powder(s). When reagentsor components are provided as a dry powder, the powder can bereconstituted by the addition of a suitable solvent. The solvent mayalso be provided in another container within the kit.

[0672] The containers of the therapeutic and diagnostic kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container or container means, into which the therapeutic andany other desired agent are placed and, preferably, suitably aliquoted.As at least two separate components are preferred, the kits willpreferably include at least two such containers. The kits may alsocomprise a third or fourth container for containing a sterile,pharmaceutically acceptable buffer or other diluent.

[0673] The kits may also contain a means by which to administer thetherapeutic agents to an animal or patient, e.g., one or more needles orsyringes, or even an eye dropper, pipette, or other such like apparatus,from which the formulations may be injected into the animal or appliedto a diseased area of the body. The kits of the present invention willalso typically include a means for containing the vials, or such like,and other component, in close confinement for commercial sale, such as,e.g., injection or blow-molded plastic containers into which the desiredvials and other apparatus are placed and retained.

[0674] L. Immunodetection and Imaging

[0675] The present invention further provides in vitro and in vivodiagnostic and imaging methods. Such methods are applicable for use ingenerating diagnostic, prognostic and/or imaging information, e.g.,related to angiogenic diseases and viral infections, and preferablyrelated to tumor treatment and imaging methods. The methods of theinvention include in vitro diagnostic tests, e.g., wherein the samplescan be obtained non-invasively and preferably tested in high throughputassays and/or where the clinical diagnosis in ambiguous and confirmationis desired. In the field of in vivo diagnostics and imaging, theantibodies and peptides of the invention are linked to one or moredetectable agents and used to form an image of an angiogenic site ortumor, optionally as a first step prior to treatment.

[0676] L1. Immunodetection Methods and Kits

[0677] The invention thus concerns immunodetection methods for binding,purifying, quantifying or otherwise generally detectingaminophospholipids and anionic phospholipids, e.g., for use indiagnosing activated and apoptotic cells and associated diseases. Theantibodies of the present invention, such as 9D2 and 3G4 (ATCC 4545),may be employed to detect aminophospholipids and anionic phospholipidsin vivo (see below), in isolated issue samples, biopsies or swabs and/orin homogenized tissue samples. Such immunodetection methods have evidentdiagnostic utility, but also have applications to non-clinical samples,such as in the titering of antigen samples, and the like.

[0678] The steps of various useful immunodetection methods have beendescribed in the scientific literature, such as, e.g., Nakamura et al.,1987, specifically incorporated herein by reference. In general, theimmunobinding methods include obtaining a sample suspected of containingaminophospholipids and/or anionic phospholipids, preferably cellssuspected of having aminophospholipids and/or anionic phospholipids atthe cell surface, and contacting the sample with an antibody of theinvention, such as 9D2 or 3G4 (ATCC 4545), under conditions effective toallow the formation of immune complexes. Any immune complexes formedduring the binding process are then detected and preferably quantified.

[0679] The sample analyzed may be a cell sample, such as cells exposedto certain test conditions in the laboratory. The sample may also be abiological sample from an animal or patient, e.g., one suspected ofhaving a disease associated with activation or apoptosis of one or morecell types. Such a sample may be a tissue section or specimen, a biopsy,a swab or smear test sample, a homogenized tissue extract or separatedor purified forms of such.

[0680] Contacting the chosen biological sample with the antibody underconditions effective and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody to the sample and incubating themixture for a period of time long enough for the antibodies to formimmune complexes with, i.e., to bind to, any aminophospholipids and/oranionic phospholipids present. After this time, the sample-antibodycomposition, such as a tissue section or ELISA plate, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those antibodies specifically bound within the primary immunecomplexes to be detected.

[0681] The detection of immunocomplex formation is well known in the artand may be achieved through the application of numerous approaches.These methods are generally based upon the detection of a label ormarker, such as any radioactive, fluorescent, biological or enzymatictags or labels known in the art. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. The use of enzymes that generate a colored product uponcontact with a chromogenic substrate are generally preferred. Secondarybinding ligands, such as a second antibody or a biotin/avidin ligandbinding arrangement, may also be used, as is known in the art.

[0682] The antibodies of the invention, such as 9D2 and 3G4 (ATCC 4545),employed in the detection may themselves be linked to a detectablelabel, wherein one would then simply detect this label, thereby allowingthe amount of the primary immune complexes in the composition to bedetermined.

[0683] Preferably, the primary immune complexes are detected by means ofa second binding ligand that has binding affinity for the antibodies ofthe invention. In such cases, the second binding ligand may be linked toa detectable label. The second binding ligand is itself often anantibody, and may thus be termed a “secondary” antibody. The primaryimmune complexes are contacted with the labeled, secondary bindingligand, or antibody, under conditions effective and for a period of timesufficient to allow the formation of secondary immune complexes. Thesecondary immune complexes are then generally washed to remove anynonspecifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

[0684] Further methods include the detection of primary immune complexesby a two step approach. A second binding ligand, such as an antibody,that has binding affinity for the first antibody is used to formsecondary immune complexes, as described above. After washing, thesecondary immune complexes are contacted with a third binding ligand orantibody that has binding affinity for the second antibody, again underconditions effective and for a period of time sufficient to allow theformation of immune complexes (tertiary immune complexes). The thirdligand or antibody is linked to a detectable label, allowing detectionof the tertiary immune complexes thus formed. This system may providefor signal amplification if desired.

[0685] Clinical diagnosis or monitoring may be applied to patients witha variety of diseases, particularly those associated with increasedaminophospholipid and/or anionic phospholipid exposure at the cellsurface. The detection of an aminophospholipid and/or anionicphospholipid, or an increase in the levels of an aminophospholipidand/or anionic phospholipid, in comparison to the levels in acorresponding biological sample from a normal subject, is indicative ofa patient with such a disease.

[0686] However, as is known to those of skill in the art, such aclinical diagnosis would not likely be made on the basis of this methodin isolation. Those of skill in the art are very familiar withdifferentiating between significant expression of a biomarker, whichrepresents a positive identification, and low level or backgroundexpression of a biomarker. Indeed, background expression levels areoften used to form a “cut-off” above which increased staining will bescored as significant or positive.

[0687] L2. In Vivo Imaging

[0688] The present invention provides a variety of in vivo diagnosticand imaging embodiments. Certain aspects of the invention concern newand surprisingly effective compositions for in vivo diagnosis andimaging. For example, any one or more of the panel of new anti-PSantibodies of the invention, preferably the 9D2 or 3G4 (ATCC 4545)antibodies or competing antibodies with like properties, are linked toan in vivo detectable agent to form an immunodiagnostic conjugate of theinvention. Although the antibodies represent an important development inthe field, the resultant immunodiagnostics may now be used in anypreviously described diagnostic or imaging embodiment connected with thedetection of an aminophospholipid and/or anionic phospholipid.

[0689] In this regard, immunodiagnostics comprising an antibody of theinvention, including the 9D2 or 3G4 (ATCC 4545) antibodies or competingantibodies with like properties, may be used in imaging vascularthromboses, particularly in or near the heart, such as in deep veinthrombosis, pulmonary embolism, myocardial infarction, atrialfibrillation, problems with prosthetic cardiovascular materials, stroke,and the like. Such compositions of the invention may also be used inimaging activated platelets, e.g., in conditions such as abscesses,restenosis, inflammation of joints and in hemostatic disorders, such asarterial, coronary, venous and cerebral thrombosis and the like. Theimmunodiagnostic compositions of the invention, preferably thosecomprising the 9D2 or 3G4 (ATCC 4545) antibodies or competing antibodieswith like properties, may also be used in detecting apoptotic cells, asmay be used in the diagnosis and imaging of a variety of diseases inwhich increased or inappropriate apoptosis occurs.

[0690] The invention further provides a range of new methods for in vivodiagnosis and imaging, which are not limited to the use of the panel ofantibodies provided herein. For example, in light of the unexpectedfinding that anionic phospholipids such as PI, PA and PG are accessibleand stably targetable markers of tumor vasculature, the inventionprovides methods for diagnosing and imaging tumors comprisingadministration of an immunodiagnostic that binds to PI, PA or PG, whichwill specifically localize to the vasculature of solid tumors. Inaddition, virally infected cells can now be detected, and viralinfections diagnosed, using an immunodiagnostic conjugate that binds toan aminophospholipid and/or an anionic phospholipid, such as PS, PE, PI,PA and PG, and preferably PS and PE.

[0691] The in vivo imaging compositions and methods of the invention canbe used in imaging per se, or in pre-imaging a site in the body to forma reliable image prior to treatment. Preferably, the imaging is tumorimaging. These compositions and methods can also be applied to imagingand diagnosis of other diseases or conditions associated withaminophospholipids and anionic phospholipids, such those involving cellactivation and/or apoptosis, including angiogenic diseases,atherosclerosis, viral infections, and other such conditions in which aninternal image is desired for diagnostic or prognostic purposes or todesign treatment.

[0692] In these embodiments, antibodies and peptides, preferably theantibodies of the invention, such as the 9D2, 3G4 (ATCC 4545) and likeantibodies, are operatively attached, linked or conjugated to adetectable label. “Detectable labels” are compounds or elements that canbe detected due to their specific functional properties, or chemicalcharacteristics, the use of which allows the component to which they areattached to be detected, and further quantified if desired. In antibodyand peptide conjugates for in vivo diagnostic protocols or “imagingmethods”, the labels can be detected using non-invasive methods.

[0693] Many appropriate imaging agents are known in the art, as aremethods for their attachment to antibodies and binding ligands (see,e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509, both incorporated hereinby reference). Certain attachment methods involve the use of a metalchelate complex employing, for example, an organic chelating agent sucha DTPA attached to the antibody (U.S. Pat. No. 4,472,509). Monoclonalantibodies may also be reacted with an enzyme in the presence of acoupling agent such as glutaraldehyde or periodate. Conjugates withfluorescein markers are prepared in the presence of these couplingagents or by reaction with an isothiocyanate.

[0694] An example of detectable labels are the paramagnetic ions. Inthis case, suitable ions include chromium (III), manganese (II), iron(III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III), withgadolinium being particularly preferred.

[0695] Ions useful in other contexts, such as X-ray imaging, include butare not limited to lanthanum (III), gold (III), lead (II), andespecially bismuth (III). Fluorescent labels include rhodamine,fluorescein and renographin. Rhodamine and fluorescein are often linkedvia an isothiocyanate intermediate.

[0696] In the case of radioactive isotopes for diagnostic applications,suitable examples include ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt,⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen, iodine¹²³, iodine¹²⁵,iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸,⁷⁵selenium, ³⁵sulphur, technetium^(99m) and yttrium⁹⁰. ¹²⁵I is oftenbeing preferred for use in certain embodiments, and technicium^(99m) andindium¹¹¹ are also often preferred due to their low energy andsuitability for long range detection.

[0697] Radioactively labeled antibodies and peptides for use in thepresent invention may be produced according to well-known methods in theart. For instance, intermediary functional groups that are often used tobind radioisotopic metallic ions to antibodies arediethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetraceticacid (EDTA).

[0698] Monoclonal antibodies can also be iodinated by contact withsodium or potassium iodide and a chemical oxidizing agent such as sodiumhypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.Anti-tumor antibodies according to the invention may be labeled withtechnetium-⁹⁹ by a ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column. Directlabeling techniques are also suitable, e.g., by incubating pertechnate,a reducing agent such as SNCl₂, a buffer solution such assodium-potassium phthalate solution, and the antibody.

[0699] Any of the foregoing type of detectably labeled antibodies andbinding ligands may be used in the imaging aspects of the invention,either for imaging alone or to form an image of a disease site or tumorprior to treatment. Either way, the methods generally compriseadministering to an animal or patient a diagnostically effective amountof an antibody or binding ligand that is conjugated to a marker that isdetectable by non-invasive methods. The antibody- or bindingligand-marker conjugate is allowed sufficient time to localize and bindto cells expressing aminophospholipids and/or anionic phospholipids inthe disease site, such as the tumor or tumor vasculature. The patient isthen exposed to a detection device to identify the detectable marker,thus forming an image of the disease site or tumor.

[0700] The nuclear magnetic spin-resonance isotopes, such as gadolinium,are detected using a nuclear magnetic imaging device; and radioactivesubstances, such as technicium^(99m) or indium¹¹¹, are detected using agamma scintillation camera or detector. U.S. Pat. No. 5,627,036 is alsospecifically incorporated herein by reference for purposes of providingeven further guidance regarding the safe and effective introduction ofdetectably labeled constructs into the blood of an individual, and meansfor determining the distribution of the detectably labeled agentextracorporally, e.g., using a gamma scintillation camera or by magneticresonance measurement.

[0701] Dosages for imaging embodiments are generally less than fortherapy, but are also dependent upon the age and weight of a patient. Aone time dose of between about 0.1, 0.5 or about 1 mg and about 9 or 10mgs, and more preferably, of between about 1 mg and about 5-10 mgs ofantibody- or binding ligand-conjugate per patient is contemplated to beuseful.

[0702] L3. Surrogate Marker for Cancer Therapy

[0703] In regard to the in vivo diagnostic and imaging, the presentinvention further provides compositions and methods for use as asurrogate marker for cancer therapy. Such embodiments concern the use ofan antibody that binds to an aminophospholipid and/or an anionicphospholipid, preferably PS, and most preferably to the use of the 9D2or 3G4 (ATCC 4545) antibodies or competing antibodies, linked to an invivo detectable agent.

[0704] Many anti-cancer therapies in current use induce apoptosis andnecrosis. Aminophospholipids and anionic phospholipids, particularly PS,are markers of pre-apoptotic and apoptotic cells. Therefore, imagingwith a suitable antibody, preferably 9D2, 3G4 (ATCC 4545) or competingantibodies, can be used to identify pre-apoptotic and apoptotic cellsand thus provide information regarding the progress of the therapy. Thisis what is meant by a “surrogate marker for cancer therapy”, as usedherein.

[0705] The use of the antibodies of the invention, preferably thosecomprising the 9D2 or 3G4 (ATCC 4545) antibodies or competing antibodieswith like properties, provides particular advantages as a surrogatemarker for cancer therapy. For example, the ability to identifypre-apoptotic cells is a particular advantage. The specificity of theantibodies will also provide more meaningful imaging data for thephysician. Also, the safety profile of these antibodies is impressiveand provides advantages over annexin, for example, as annexin suffersfrom drawbacks associated with coagulation.

[0706] Accordingly, any of the in vivo diagnostic and imaging methodsdescribed above may be adapted for prognostic use as a surrogate markerfor cancer therapy simply by use in a patient undergoing cancer therapy.

[0707] M. Tumor Treatment

[0708] Important aspects of the present invention concern the treatmentof malignancies, tumors and vascularized tumors. This includes tumors inwhich angiogenesis is more or less important and tumors havingprothrombotic blood vessels. The treatment of benign tumors is includedin the invention, such as acoustic neuroma, neurofibroma, trachoma,pyogenic granulomas and BPH. The treatment of blood-born tumors, such asleukemias, and various acute or chronic neoplastic diseases of the bonemarrow is also encompassed.

[0709] The present invention is broadly applicable to the treatment ofany malignant tumor, whether having a vascular component or not. Tumorsfor treatment include solid tumors, particularly carcinomas, whichrequire a vascular component for the provision of oxygen and nutrients.Exemplary solid tumors that may be treated using the invention include,but are not limited to, carcinomas of the lung, breast, ovary, stomach,pancreas, larynx, esophagus, testes, liver, parotid, biliary tract,colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate,thyroid, squamous cell carcinomas, adenocarcinomas, small cellcarcinomas, melanomas, gliomas, glioblastomas, neuroblastomas, and thelike.

[0710] The present invention is contemplated for use in the treatment ofany patient that presents with a solid tumor. In general, the inventioncan be used to treat tumors of all sizes, including those about 0.3-0.5cm and upwards, tumors of greater than 0.5 cm in size and patientspresenting with tumors of between about 1.0 and about 2.0 cm in size,although tumors up to and including the largest tumors found in humansmay also be treated.

[0711] Although the present invention is not generally intended as apreventative or prophylactic treatment, use of the invention iscertainly not confined to the treatment of patients having tumors ofonly moderate or large sizes. There are many reasons underlying theseaspects of the invention. For example, a patient presenting with aprimary tumor of moderate size or above may also have various othermetastatic tumors that are considered to be small-sized or even in theearlier stages of metastatic tumor seeding. Given that theanti-aminophospholipid or anti-anionic phospholipid antibodies orPE-binding peptide derivatives, or combinations, of the invention aregenerally administered into the systemic circulation of a patient, theywill naturally have effects on the secondary, smaller and metastatictumors, although this may not be the primary intent of the treatment.Furthermore, even in situations where the tumor mass as a whole is asingle small tumor, certain beneficial anti-tumor effects will resultfrom the use of the present treatments.

[0712] The guidance provided herein regarding the suitable patients foruse in connection with the present invention is intended as teachingthat certain patient's profiles may assist with the selection ofpatients for treatment by the present invention. The pre-selection ofcertain patients, or categories of patients, does not in any way negatethe basic usefulness of the present invention in connection with thetreatment of all patients having cancer. A further consideration is thefact that the assault on the tumor provided by the antibody therapy ofthe invention may predispose the tumor to further therapeutic treatment,such that the subsequent treatment results in an overall synergisticeffect or even leads to total remission or cure.

[0713] It is not believed that any particular type of tumor should beexcluded from treatment using the present invention. However, the typeof tumor cells may be relevant to the use of the invention incombination with tertiary therapeutic agents, particularlychemotherapeutics and anti-tumor cell immunotoxins. As the presentinvention includes within its modes of action the targeting anddestruction of tumor vasculature, and as the vasculature issubstantially or entirely the same in all solid tumors, it will beunderstood that the present methodology is widely or entirely applicableto the treatment of all solid tumors, irrespective of the particularphenotype or genotype of the tumor cells themselves. The data presentedherein is compelling as it shows impressive results in a wide range ofdifferent tumor models.

[0714] Therapeutically effective doses are readily determinable usingdata from an animal model, as shown in the studies detailed herein, andfrom clinical data using a range of therapeutic agents. Experimentalanimals bearing solid tumors are frequently used to optimize appropriatetherapeutic doses prior to translating to a clinical environment. Suchmodels are known to be very reliable in predicting effective anti-cancerstrategies. For example, mice bearing solid tumors, such as used in theExamples, are widely used in pre-clinical testing. The inventors haveused such art-accepted mouse models to determine working ranges oftherapeutic agents that give beneficial anti-tumor effects with minimaltoxicity.

[0715] In terms of tumor therapy, bearing in mind the attendant safetybenefits associated with the overall invention, one may refer to thescientific and patent literature on the success of using otheranti-vascular therapies. By way of example, U.S. Pat. Nos. 5,855,866;5,877,289; 5,965,132; 6,051,230; 6,004,555; 5,776,427; 6,004,554;6,036,955; and 6,093,399 are incorporated herein by reference for thepurpose of further describing the use of such agents as may be appliedto those of the present invention. U.S. Pat. Nos. 6,312,694 and6,406,693 are further specifically incorporated herein by reference forguidance on dosing and treatment using unconjugated antibodies to PS andPE and related immunoconjugates.

[0716] As is known in the art, there are realistic objectives that maybe used as a guideline in connection with pre-clinical testing beforeproceeding to clinical treatment. However, due to the safety alreadydemonstrated in accepted models, pre-clinical testing of the presentinvention will be more a matter of optimization, rather than to confirmeffectiveness. Thus, pre-clinical testing may be employed to select themost advantageous agents, doses or combinations.

[0717] Any antibody dose, combined method or medicament that results inany consistently detectable anti-tumor effect, including detectabletumor vasculature regression, thrombosis and/or destruction and tumornecrosis, will still define a useful invention. Regressive, thrombotic,destructive and necrotic effects should preferably be observed inbetween about 10% and about 40-50% of the tumor blood vessels and tumortissues, upwards to between about 50% and about 99% of such effectsbeing observed. The present invention may also be effective againstvessels downstream of the tumor, i.e., target at least a sub-set of thedraining vessels, particularly as cytokines released from the tumor willbe acting on these vessels, changing their antigenic profile.

[0718] It will also be understood that even in such circumstances wherethe anti-tumor effects of the therapy are towards the low end of thisrange, it may be that this therapy is still equally or even moreeffective than all other known therapies in the context of theparticular tumor. It is unfortunately evident to a clinician thatcertain tumors cannot be effectively treated in the intermediate or longterm, but that does not negate the usefulness of the present therapy,particularly where it is at least about as effective as the otherstrategies generally proposed.

[0719] In designing appropriate doses of anti-aminophospholipid oranti-anionic phospholipid antibodies, PE-binding peptide derivatives orcombined therapeutics for the treatment of vascularized tumors, one mayreadily extrapolate from the animal studies described herein in order toarrive at appropriate doses for clinical administration. To achieve thisconversion, one would account for the mass of the agents administeredper unit mass of the experimental animal and, preferably, account forthe differences in the body surface area between the experimental animaland the human patient. All such calculations are well known and routineto those of ordinary skill in the art.

[0720] For example, in taking the successful doses of therapeutics usedin the mouse studies, and applying standard calculations based upon massand surface area, effective doses of agents for use in human patientswould be between about 1 mg and about 500 mgs antibody per patient, andpreferably, between about 10 mgs and about 100 mgs antibody per patient.

[0721] Accordingly, using this information, the inventors contemplatethat useful low doses for human administration will be about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25 or about 30 mgs or so per patient; anduseful high doses for human administration will be about 250, 275, 300,325, 350, 375, 400, 425, 450, 475 or about 500 mgs or so per patient.Useful intermediate doses for human administration are contemplated tobe about 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or about225 mgs or so per patient. In general, dosage ranges of between about5-100 mgs, about 10-80 mgs, about 20-70 mgs, about 25-60 mgs, or about30-50 mgs per patient will be preferred. However, any particular rangeusing any of the foregoing recited exemplary doses or any valueintermediate between the particular stated ranges is contemplated.

[0722] Notwithstanding the stated ranges, it will be understood that,given the parameters and detailed guidance presented herein, furthervariations in the active or optimal ranges will be encompassed withinthe present invention. It will thus be understood that lower doses maybe more appropriate in combination with certain agents, and that highdoses can still be tolerated, particularly given the enhanced safety ofthe present constructs. The use of human or humanized antibodies andhuman effectors renders the present invention even safer for clinicaluse, further reducing the chances of significant toxicity or sideeffects in healthy tissues.

[0723] The intention of the therapeutic regimens of the presentinvention is generally to produce significant anti-tumor effects whilststill keeping the dose below the levels associated with unacceptabletoxicity. In addition to varying the dose itself, the administrationregimen can also be adapted to optimize the treatment strategy. Acurrently preferred treatment strategy is to administer between about1-500 mgs, and preferably, between about 10-100 mgs of the antibody, ortherapeutic cocktail containing such, about 3 times within about a 7 dayperiod. For example, doses would be given on about day 1, day 3 or 4 andday 6 or 7.

[0724] In administering the particular doses themselves, one wouldpreferably provide a pharmaceutically acceptable composition (accordingto FDA standards of sterility, pyrogenicity, purity and general safety)to the patient systemically. Intravenous injection is generallypreferred, and the most preferred method is to employ a continuousinfusion over a time period of about 1 or 2 hours or so. Although it isnot required to determine such parameters prior to treatment using thepresent invention, it should be noted that the studies detailed hereinresult in at least some thrombosis being observed specifically in theblood vessels of a solid tumor within about 12-24 hours of injection,and that the tumor cells themselves begin to die within about 24 to 72hours. Widespread tumor necrosis is generally observed in the next about48-96 hours, up to and including greater than 60% necrosis beingobserved.

[0725] Naturally, before wide-spread use, clinical trials will beconducted. The various elements of conducting a clinical trial,including patient treatment and monitoring, will be known to those ofskill in the art in light of the present disclosure. The followinginformation is being presented as a general guideline for use inestablishing such trials.

[0726] Patients chosen for the first treatment studies will have failedto respond to at least one course of conventional therapy, and will haveobjectively measurable disease as determined by physical examination,laboratory techniques, and/or radiographic procedures. Any chemotherapyshould be stopped at least 2 weeks before entry into the study. Wheremurine monoclonal antibodies or antibody portions are employed, thepatients should have no history of allergy to mouse immunoglobulin.

[0727] Certain advantages will be found in the use of an indwellingcentral venous catheter with a triple lumen port. The therapeuticsshould be filtered, for example, using a 0.22μ filter, and dilutedappropriately, such as with saline, to a final volume of 100 ml. Beforeuse, the test sample should also be filtered in a similar manner, andits concentration assessed before and after filtration by determiningthe A₂₈₀. The expected recovery should be within the range of 87% to99%, and adjustments for protein loss can then be accounted for.

[0728] The constructs may be administered over a period of approximately4-24 hours, with each patient receiving 2-4 infusions at 2-7 dayintervals. Administration can also be performed by a steady rate ofinfusion over a 7 day period. The infusion given at any dose levelshould be dependent upon any toxicity observed. Hence, if Grade IItoxicity was reached after any single infusion, or at a particularperiod of time for a steady rate infusion, further doses should bewithheld or the steady rate infusion stopped unless toxicity improved.Increasing doses should be administered to groups of patients untilapproximately 60% of patients showed unacceptable Grade III or IVtoxicity in any category. Doses that are 2/3 of this value are definedas the safe dose.

[0729] Physical examination, tumor measurements, and laboratory testsshould, of course, be performed before treatment and at intervals up to1 month later. Laboratory tests should include complete blood counts,serum creatinine, creatine kinase, electrolytes, urea, nitrogen, SGOT,bilirubin, albumin, and total serum protein. Serum samples taken up to60 days after treatment should be evaluated by radioimmunoassay for thepresence of the administered construct, and antibodies against anyportions thereof. Immunological analyses of sera, using any standardassay such as, for example, an ELISA or RIA, will allow thepharmacokinetics and clearance of the therapeutics to be evaluated.

[0730] To evaluate the anti-tumor responses, the patients should beexamined at 48 hours to 1 week and again at 30 days after the lastinfusion. When palpable disease was present, two perpendicular diametersof all masses should be measured daily during treatment, within 1 weekafter completion of therapy, and at 30 days. To measure nonpalpabledisease, serial CT scans could be performed at 1-cm intervals throughoutthe chest, abdomen, and pelvis at 48 hours to 1 week and again at 30days. Tissue samples should also be evaluated histologically, and/or byflow cytometry, using biopsies from the disease sites or even blood orfluid samples if appropriate.

[0731] Clinical responses may be defined by acceptable measure. Forexample, a complete response may be defined by the disappearance of allmeasurable tumor 1 month after treatment. Whereas a partial response maybe defined by a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules 1 month aftertreatment, with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater 1 month aftertreatment, with progression in one or more sites.

[0732] In light of results from clinical trials, such as those describedabove, an even more precise treatment regimen may be formulated. Evenso, some variation in dosage may later be necessary depending on thecondition of the subject being treated. The physician responsible foradministration will, in light of the present disclosure, be able todetermine the appropriate dose for the individual subject. Suchoptimization and adjustment is routinely carried out in the art, and byno means reflects an undue amount of experimentation.

[0733] N. Combination Tumor Therapies

[0734] The treatment methods of the present invention may be combinedwith any other methods generally employed in the treatment of theparticular tumor, disease or disorder that the patient exhibits. So longas a particular therapeutic approach is not known to be detrimental tothe patient's condition in itself, and does not significantly counteractthe anti-aminophospholipid or anti-anionic phospholipid-based treatmentof the invention, its combination with the present invention iscontemplated.

[0735] Combination therapy for non malignant diseases is alsocontemplated. A particular example of such is benign prostatichyperplasia (BPH), which may be treated in combination other treatmentscurrently practiced in the art. For example, targeting of immunotoxinsto markers localized within BPH, such as PSA.

[0736] In connection solid tumor treatment, the present invention may beused in combination with classical approaches, such as surgery,chemotherapy, radiotherapy, cytokine therapy, anti-angiogenesis and thelike. The invention therefore provides combined therapies in which theantibodies, immunoconjugates or peptide conjugates are usedsimultaneously with, before, or after surgery or radiation treatment; orare administered to patients with, before, or after conventionalchemotherapeutic or radiotherapeutic agents, cytokines, anti-angiogenicagents, apoptosis-inducing agents, targeted immunotoxins or coaguligandsor such like. Many examples of suitable therapeutic agents have beendescribed above in connection with the immunoconjugate aspects of thepresent invention. Any of the agents initially described for use as onepart of a therapeutic conjugate may also be used separately, in thecombination therapies of the present invention.

[0737] In terms of surgery, any surgical intervention may be practicedin combination with the present invention. In connection withradiotherapy, any mechanism for inducing DNA damage locally within tumorcells is contemplated, such as γ-irradiation, X-rays, UV-irradiation,microwaves and even electronic emissions and the like. The directeddelivery of radioisotopes to tumor cells is also contemplated, and thismay be used in connection with a targeting antibody or other targetingmeans.

[0738] The general use of combinations of substances in cancer treatmentis well known. For example, U.S. Pat. No. 5,710,134 (incorporated hereinby reference) discloses components that induce necrosis in tumors incombination with non-toxic substances or “prodrugs”. The enzymes setfree by necrotic processes cleave the non-toxic “prodrug” into the toxic“drug”, which leads to tumor cell death. Also, U.S. Pat. No. 5,747,469(incorporated herein by reference) discloses the combined use of viralvectors encoding p53 and DNA damaging agents. Any such similarapproaches can be used with the present invention.

[0739] When one or more agents are used in combination with theantibodies, immunoconjugates and peptide-based therapeutics of thepresent invention, there is no requirement for the combined results tobe additive of the effects observed when each treatment is conductedseparately. Although at least additive effects are generally desirable,any increased anti-tumor effect above one of the single therapies wouldbe of benefit. Also, there is no particular requirement for the combinedtreatment to exhibit synergistic effects, although this is certainlypossible and advantageous.

[0740] N1. Selection of Second Anti-Cancer Agents

[0741] The “primary therapeutic agents” of the present invention, asused herein, are anti-aminophospholipid or anti-anionic phospholipidantibodies, immunoconjugates or PE-binding peptide derivatives andconjugates. The “secondary therapeutic agents”, as used herein, aresecond, distinct therapeutic agents or anti-cancer agents, i.e.,therapeutic agents or anti-cancer agents “other than” the primarytherapeutic agent. Any secondary therapeutic agent may be used in thecombination therapies of the present invention. Also, secondarytherapeutic agents or “second anti-cancer agents” may be selected with aview to achieving additive, greater than additive and potentiallysynergistic effects, according to the following guidance.

[0742] To practice combined anti-tumor therapy, one would simplyadminister to an animal or patient an anti-aminophospholipid oranti-anionic phospholipid antibody, immunoconjugate or PE-bindingpeptide-based therapeutic of the present invention in combination withanother, i.e., a second, distinct anti-cancer agent in a mannereffective to result in their combined anti-tumor actions within theanimal or patient. The agents would therefore be provided in amountseffective and for periods of time effective to result in their combinedpresence within the tumor or tumor vasculature and their combinedactions in the tumor environment. To achieve this goal, the primarytherapeutics of the present invention and the second, distinctanti-cancer agents may be administered to the animal substantiallysimultaneously, either in a single composition, or as two distinctcompositions using different administration routes.

[0743] Alternatively, the anti-aminophospholipid or anti-anionicphospholipid antibody, immunoconjugate or PE-binding peptide-basedtherapeutic of the present invention may precede, or follow, the second,distinct anti-cancer agent by, e.g., intervals ranging from minutes toweeks. In certain embodiments where the primary therapeutics of thepresent invention and the second, distinct anti-cancer agents areapplied separately to the animal, one would ensure that a significantperiod of time did not expire between the time of each delivery, suchthat each agent would still be able to exert an advantageously combinedeffect on the tumor. In such instances, it is contemplated that onewould contact the tumor with both agents within about 5 minutes to aboutone week of each other and, more preferably, within about 12-72 hours ofeach other, with a delay time of only about 12-48 hours being mostpreferred.

[0744] The secondary therapeutic agents for separately timed combinationtherapies may be selected based upon certain criteria, including thosediscussed below. However, a preference for selecting one or more second,distinct anti-cancer agents for prior or subsequent administration doesnot preclude their use in substantially simultaneous administration ifdesired.

[0745] Second, distinct anti-cancer agents selected for administration“prior to” the primary therapeutic agents of the present invention, anddesigned to achieve increased and potentially synergistic effects,include agents that induce the expression of aminophospholipids oranionic phospholipids within the tumor vasculature. For example, agentsthat stimulate localized calcium production, activate membranetransporters that move PS and other phospholipids to the outer surfaceof the plasma membrane, injure the tumor endothelium, cause preapoptoticchanges and/or induce apoptosis in the tumor endothelium will generallyresult in increased aminophospholipid and anionic phospholipidexpression. Examples of such agents are docetaxel and paclitaxol. Theaminophospholipids and anionic phospholipids can then be targeted usingan antibody of the invention, thus amplifying the overall therapeuticeffect, and also giving increased attack via host effectors (complement,ADCC, antibody-mediated phagocytosis, CDC).

[0746] Drugs that have selectivity for angiogenic, remodeling oractivated endothelial cells, such as are present in tumor blood vessels,but not in normal resting blood vessels, can also be used to selectivelycauses exposure of PS and other phospholipids on the surface of tumorendothelial cells. Examples of such agents are combretastatins anddocetaxel. This again would lead to increased antibody binding andenhanced initiation of host effector mechanisms.

[0747] Second, distinct anti-cancer agents selected for administration“subsequent to” the primary therapeutic agents of the present invention,and designed to achieve increased and potentially synergistic effects,include agents that benefit from the effects of the primary therapeuticagent. The anti-aminophospholipid or anti-anionic phospholipid antibody,immunoconjugate or peptide-based therapeutic of the present inventionwill cause tumor destruction. Accordingly, effective second, distinctanti-cancer agents for subsequent administration include anti-angiogenicagents, which inhibit metastasis; agents targeting necrotic tumor cells,such as antibodies specific for intracellular antigens that becomeaccessible from malignant cells in vivo (U.S. Pat. Nos. 5,019,368,4,861,581 and 5,882,626, each specifically incorporated herein byreference); and chemotherapeutic agents and anti-tumor cellimmunoconjugates, which attack any tumor cells that may survive at theperiphery.

[0748] In some situations, it may be desirable to extend the time periodfor treatment significantly, where several days (2, 3, 4, 5, 6 or 7),several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even several months (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Thiswould be advantageous in circumstances where one treatment was intendedto substantially destroy the tumor, such as the primary therapeuticagent of the present invention, and another treatment was intended toprevent micrometastasis or tumor re-growth, such as the administrationof an anti-angiogenic agent. Anti-angiogenics should be administered ata careful time after surgery, however, to allow effective wound healing.Anti-angiogenic agents may then be administered for the lifetime of thepatient.

[0749] It is also envisioned that more than one administration of eitherthe primary therapeutic agent or the second, distinct anti-cancer agentwill be utilized. The primary therapeutic agent and the second, distinctanti-cancer agent may be administered interchangeably, on alternate daysor weeks; or a sequence of one agent treatment may be given, followed bya sequence of the other treatment. In any event, to achieve tumorregression using a combined therapy, all that is required is to deliverboth agents in a combined amount effective to exert an anti-tumoreffect, irrespective of the times for administration.

[0750] Whether administered substantially simultaneously orsequentially, the anti-aminophospholipid and anti-anionic phospholipidantibodies and therapeutics of the present invention may be administeredin combination with one or more chemotherapeutic agents or drugs.Chemotherapeutic drugs can kill proliferating tumor cells, enhancing thenecrotic areas created by the overall treatment. The drugs can thusenhance the thrombotic action of the primary therapeutic agents of theinvention.

[0751] Most cancer chemotherapeutic drugs are selective for dividing,oxygenated cells. These have advantages in combined therapy as thechemotherapeutic drug acts on different targets from the primarytherapeutic agents of the invention, leading to a more completeanti-vascular or anti-tumor effect. For example, chemotherapeutic drugsare selectively active against the rapidly dividing, oxygenated tumorcells in the tumor periphery, whereas the agents of the invention actprimarily on vessels or tumor cells in the ‘stressed’ tumor core, whereactivating reactive oxygen species are abundant. Anti-angiogenic drugsthat are selective for well-oxygenated, angiogenic vessels in the tumorperiphery would also be effective in combination, as the agents of theinvention act on the relatively hypoxic, quiescent vessels in the tumorcore.

[0752] By inducing the formation of thrombi in tumor vessels, theprimary therapeutic agents of the present invention can also enhance theaction of the chemotherapeutic drugs by retaining or trapping the drugswithin the tumor. The chemotherapeutics are thus retained within thetumor, while the rest of the drug is cleared from the body. Tumor cellsare thus exposed to a higher concentration of drug for a longer periodof time. This entrapment of drug within the tumor makes it possible toreduce the dose of drug, making the treatment safer as well as moreeffective.

[0753] Further drugs for combined use in the present invention are thosethat act on cells that are “sensitized” to the drug by the action of theprimary therapeutic agent, such that reduced doses of the second drugare needed to achieve its anti-tumor effect. For example, this couldoccur where a major component of the second drug's action is exerted ontumor vessels and the antibodies or agents of the invention sensitizethe cells to the drug. The same is true where the primary therapeuticagent of the invention sensitizes tumor cells to a second drug, eitherdirectly or through stimulation of cytokine release.

[0754] Other suitable second anti-cancer agents for combination therapyare those that enhance the activity of host effector cells, e.g., byselectively inhibiting the activity of immunosuppressive components ofthe immune system. Such agents enable the primary therapeutic agents ofthe invention, which stimulate attack by effector cells as part of theirmechanism, to work more aggressively. An example of such an agent isdocetaxel.

[0755] Although an understanding of the precise mechanism(s) of actionof the primary therapeutic agents is not necessary to practice thetreatment of the invention, data and reasoned deductions concerning suchmechanisms can be used to select particular second anti-cancer agentsfor combined use in the present invention. The effectiveness of thechosen combination therapy, in turn, supports the original data andproposed mechanisms of action, and also leads to preferred categories ofsecond anti-cancer agents for practicing combination therapy.

[0756] Drugs that induce apoptosis are preferred for use in thecombination therapies. Docetaxel, for example, induces apoptosis andtherefore PS exposure by binding to microtubules and disrupting cellmitosis (Hotchkiss et al., 2002). Treatment of endothelial cells, whichline tumor blood vessels, and tumor cells with docetaxel at subclinicalconcentrations is herein shown to induce PS expression at the cellsurface, as demonstrated by strong binding of the 3G4 antibody in vitro.

[0757] The present inventors have also determined that the anti-tumoreffects of the antibodies of the invention include Fc domain-mediatedaugmentation of immune effector functions, as shown by increasedantibody-mediated phagocytosis. Therefore, the antibodies should alsoexert other Fc domain-mediated functions, such as ADCC, CDC, stimulationof cytokine production, and such mechanisms in combination. This is alsorelevant to docetaxel, as other studies have shown that the treatment ofbreast cancer patients with docetaxel leads to increases in serum IFN-γ,IL-2, IL-6 and GM-CSF cytokine levels, augmenting the anti-tumor immuneresponses in these patients by enhancing the activity of natural killer(NK) and lymphokine activated killer (LAK) cells (Tsavaris et al.,2002).

[0758] Therefore, the inventors reasoned that docetaxel will both inducePS expression and binding of the administered antibody, and alsoenhances the activities of immune effectors, which mediate anti-tumoreffects. Based upon the foregoing considerations, the inventors haveshown that combination of the antibodies of the present invention, asexemplified by the 3G4 antibody, with docetaxel was significantlysuperior to either docetaxel or 3G4 alone in mice bearing orthotopicMDA-MB-435 human breast cancer xenografts (Example XX).

[0759] Accordingly, docetaxel and other chemotherapeutic agents thatinduce apoptosis are preferred agents for use in the combinationtreatments of the present invention. Combinations of antibodies toaminophospholipids and/or anionic phospholipids with chemotherapeuticsdrugs that induce apoptosis, such as docetaxel, should synergisticallyattack tumor vasculature endothelial cell and tumor cell compartments,leading to not only significantly enhanced treatment efficacy but alsolower toxicity. These combinations are contemplated for use in breastcancer treatment, particularly the combination of metronomicchemotherapy using docetaxel with an antibody of the present invention.

[0760] N2. Endotoxin

[0761] Endotoxin and detoxified endotoxin derivatives may be used in thecombination treatment, preferably at low doses (PCT Publication No. WO03/028840, specifically incorporated herein by reference). Variousdetoxified endotoxins are available, which are preferred for use inanimals and particularly for use in humans. Detoxified and refinedendotoxins, and combinations thereof, are described in U.S. Pat. Nos.4,866,034; 4,435,386; 4,505,899; 4,436,727; 4,436,728; 4,505,900, eachspecifically incorporated herein by reference.

[0762] The non-toxic derivative monophosphoryl lipid A (MPL) is oneexample of a detoxified endotoxin that may be used in the presentinvention. MPL is known to be safe for humans; clinical trials using MPLas an adjuvant have shown 100 μg/m² to be safe for human use, even on anoutpatient basis.

[0763] N3. Cytokines

[0764] Cytokine therapy has proven to be an effective partner forcombined therapeutic regimens. Various cytokines may be employed in thecombined approaches of the present invention. Examples of cytokinesinclude IL-1α IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, TGF-β, GM-CSF, M-CSF, G-CSF, TNFα, TNFβ,LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β,IFN-γ. Cytokines are administered according to standard regimens,consistent with clinical indications such as the condition of thepatient and relative toxicity of the cytokine. Uteroglobins may also beused to prevent or inhibit metastases (U.S. Pat. No. 5,696,092;incorporated herein by reference).

[0765] N4. TNFα and Inducers of TNFα

[0766] TNFα and inducers of TNFα may also be used in combination withthe present invention. TNFα increases vascular permeability, and istherefore useful in facilitating the penetration of anti-cancer agentsinto the tumor. Although antibody localization is by no means a problemwhen targeting aminophospholipid and anionic phospholipids, as in thepresent invention, the combined use of TNFα can facilitate access ofother chemotherapeutics and immunoconjugates to the tumor, and evenincrease binding of the antibodies of the invention to far distant tumorcells.

[0767] Low levels of endotoxin, Rac1 antagonists, such as an attenuatedor engineered adenovirus, DMXAA (and FAA), CM101 and thalidomide mayalso be used. Rac1 antagonists may be used in the combined treatment ofthe present invention, as about 5000 DNA particles per cell cause TNFupregulation independent of CD14 (Sanlioglu et al., 2001). CM101,thalidomide and DMXAA can also be used in combination herewith, atstandard or reduced doses.

[0768] N5. Chemotherapeutics

[0769] Irrespective of the underlying mechanism(s), a variety ofchemotherapeutic agents may be used in the combined treatment methodsdisclosed herein. Chemotherapeutic agents contemplated for combined useinclude, e.g., tamoxifen, taxol, vinblastine, etoposide (VP-16),adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D, mitomycinC, combretastatin(s), more particularly docetaxel (taxotere), cisplatin(CDDP), cyclophosphamide, doxorubicin, methotrexate, paclitaxel andvincristine, and derivatives and prodrugs thereof.

[0770] As will be understood by those of ordinary skill in the art,appropriate doses of chemotherapeutic agents include those alreadyemployed in clinical therapies wherein the chemotherapeutics areadministered alone or in combination with other chemotherapeutics.However, lower doses are now possible due to the advantages provided bythe present invention. By way of example only, agents such as cisplatin,and other DNA alkylating may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

[0771] Further useful agents include compounds that interfere with DNAreplication, mitosis, chromosomal segregation and/or tubulin activity.Such chemotherapeutic compounds include adriamycin, also known asdoxorubicin, etoposide, verapamil, podophyllotoxin(s), combretastatin(s)and the like. Widely used in a clinical setting for the treatment ofneoplasms, these compounds are administered through bolus injectionsintravenously at doses ranging from 25-75 mg/m² at 21 day intervals foradriamycin, to 35-50 mg/m² for etoposide intravenously or double theintravenous dose orally.

[0772] Agents that disrupt the synthesis and fidelity of polynucleotideprecursors may also be used. Particularly useful are agents that haveundergone extensive testing and are readily available. As such, agentssuch as 5-fluorouracil (5-FU) are preferentially used by neoplastictissue, making this agent particularly useful for targeting toneoplastic cells. Although quite toxic, 5-FU, is applicable in a widerange of carriers, including topical, however intravenous administrationwith doses ranging from 3 to 15 mg/kg/day being commonly used.

[0773] Exemplary chemotherapeutic agents that are useful in connectionwith combined therapy are listed in Table D. Each of the agents listedtherein are exemplary and by no means limiting. The skilled artisan isdirected to “Remington's Pharmaceutical Sciences” 15th Edition, chapter33, in particular pages 624-652. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The physician responsible for administration will be able todetermine the appropriate dose for the individual subject. TABLE DCHEMOTHERAPEUTIC AGENTS USEFUL IN NEOPLASTIC DISEASE NONPROPRIETARYNAMES CLASS TYPE OF AGENT (OTHER NAMES) DISEASE Alkylating AgentsNitrogen Mustards Mechlorethamine (HN₂) Hodgkin's disease, non-Hodgkin'slymphomas Cyclophosphamide Acute and chronic lymphocytic Ifosfamideleukemias, Hodgkin's disease, non- Hodgkin's lymphomas, multiplemyeloma, neuroblastoma, breast, ovary, lung, Wilms' tumor, cervix,testis, soft-tissue sarcomas Melphalan (L-sarcolysin) Multiple myeloma,breast, ovary Chlorambucil Chronic lymphocytic leukemia, primarymacroglobulinemia, Hodgkin's disease, non-Hodgkin's lymphomasEthylenimenes and Hexamethylmelamine Ovary Methylmelamines ThiotepaBladder, breast, ovary Alkyl Sulfonates Busulfan Chronic granulocyticleukemia Nitrosoureas Carmustine (BCNU) Hodgkin's disease, non-Hodgkin'slymphomas, primary brain tumors, multiple myeloma, malignant melanomaLomustine (CCNU) Hodgkin's disease, non-Hodgkin's lymphomas, primarybrain tumors, small-cell lung Semustine (methyl-CCNU) Primary braintumors, stomach, colon Streptozocin Malignant pancreatic insulinoma,(streptozotocin) malignant carcinoid Triazines Dacarbazine (DTIC;Malignant melanoma, Hodgkin's dimethyltriazenoimidaz disease,soft-tissue sarcomas olecarboxamide) Antimetabolites Folic Acid AnalogsMethotrexate Acute lymphocytic leukemia, (amethopterin) choriocarcinoma,mycosis fungoides, breast, head and neck, lung, osteogenic sarcomaPyrimidine Analogs Fluouracil (5-fluorouracil; Breast, colon, stomach,pancreas, 5-FU) ovary, head and neck, urinary bladder, Floxuridine(fluorode- premalignant skin lesions (topical) oxyuridine; FUdR)Cytarabine (cytosine Acute granulocytic and acute arabinoside)lymphocytic leukemias Mercaptopurine Acute lymphocytic, acutegranulocytic (6-mercaptopurine; and chronic granulocytic leukemias 6-MP)Purine Analogs and Thioguanine Acute granulocytic, acute lymphocyticRelated Inhibitors (6-thioguanine; TG) and chronic granulocyticleukemias Pentostatin Hairy cell leukemia, mycosis (2-deoxycoformycin)fungoides, chronic lymphocytic leukemia Natural Products Vinca AlkaloidsVinblastine (VLB) Hodgkin's disease, non-Hodgkin's lymphomas, breast,testis Vincristine Acute lymphocytic leukemia, neuroblastoma, Wilms'tumor, rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin's lymphomas,small-cell lung Epipodophyllotoxins Etoposide Testis, small-cell lungand other lung, Tertiposide breast, Hodgkin's disease, non- Hodgkin'slymphomas, acute granulocytic leukemia, Kaposi's sarcoma AntibioticsDactinomycin Choriocarcinoma, Wilms' tumor, (actinomycin D)rhabdomyosarcoma, testis, Kaposi's sarcoma Daunorubicin Acutegranulocytic and acute (daunomycin; lymphocytic leukemias rubidomycin)Doxorubicin Soft-tissue, osteogenic and other sarcomas; Hodgkin'sdisease, non- Hodgkin's lymphomas, acute leukemias, breast,genitourinary, thyroid, lung, stomach, neuroblastoma Bleomycin Testis,head and neck, skin, esophagus, lung and genitourinary tract; Hodgkin'sdisease, non- Hodgkin's lymphomas Plicamycin (mithramycin) Testis,malignant hypercalcemia Mitomycin (mitomycin C) Stomach, cervix, colon,breast, pancreas, bladder, head and neck Enzymes L-Asparaginase Acutelymphocytic leukemia Biological Response Interferon alfa Hairy cellleukemia., Kaposi's Modifiers sarcoma, melanoma, carcinoid, renal cell,ovary, bladder, non-Hodgkin's lymphomas, mycosis fungoides, multiplemyeloma, chronic granulocytic leukemia Miscellaneous PlatinumCoordination Cisplatin (cis-DDP) Testis, ovary, bladder, head and neck,Agents Complexes Carboplatin lung, thyroid, cervix, endometrium,neuroblastoma, osteogenic sarcoma Anthracenedione Mitoxantrone Acutegranulocytic leukemia, breast Substituted Urea Hydroxyurea Chronicgranulocytic leukemia, polycythemia vera, essental thrombocytosis,malignant melanoma Methyl Hydrazine Procarbazine Hodgkin's diseaseDerivative (N-methylhydrazine, MIH) Adrenocortical Mitotane (o, p′-DDD)Adrenal cortex Suppressant Aminoglutethimide Breast Hormones andAdrenocorticosteroids Prednisone (several other Acute and chroniclymphocytic Antagonists equivalent preparations leukemias, non-Hodgkin'slymphomas, available) Hodgkin's disease, breast ProgestinsHydroxyprogesterone Endometrium, breast caproate Medroxyprogesteroneacetate Megestrol acetate Estrogens Diethylstilbestrol Breast, prostateEthinyl estradiol (other preparations available) Antiestrogen TamoxifenBreast Androgens Testosterone propionate Breast Fluoxymesterone (otherpreparations available) Antiandrogen Flutamide ProstateGonadotropin-releasing Leuprolide Prostate hormone analog

[0774] N6. Anti-Angiogenics

[0775] The term “angiogenesis” refers to the generation of new bloodvessels, generally into a tissue or organ. Under normal physiologicalconditions, humans or animals undergo angiogenesis only in specificrestricted situations. For example, angiogenesis is normally observed inwound healing, fetal and embryonic development and formation of thecorpus luteum, endometrium and placenta. New evidence, however, showsthat angiogenesis is important in certain normal situations, such as inadrenal tissue, prostate and ovary. The therapeutic agents of thepresent invention, in which anti-angiogenesis is not the only mode ofaction, thus have advantages over prominent anti-angiogenic therapies,such as antibody A4.6.1 (Brem, 1998; Baca et al., 1997; Presta et al.,1997), in that desirable or “physiological” angiogenesis will not beinhibited when using the present invention.

[0776] Uncontrolled (persistent and/or unregulated) angiogenesis isrelated to various disease states, and occurs during tumor developmentand metastasis. Both controlled and uncontrolled angiogenesis arethought to proceed in a similar manner. Endothelial cells and pericytes,surrounded by a basement membrane, form capillary blood vessels.Angiogenesis begins with the erosion of the basement membrane by enzymesreleased by endothelial cells and leukocytes. The endothelial cells,which line the lumen of blood vessels, then protrude through thebasement membrane. Angiogenic stimulants induce the endothelial cells tomigrate through the eroded basement membrane. The migrating cells form a“sprout” off the parent blood vessel, where the endothelial cellsundergo mitosis and proliferate. The endothelial sprouts merge with eachother to form capillary loops, creating the new blood vessel.

[0777] Despite the new evidence that angiogenesis is required in somenormal tissues, anti-angiogenic therapies are still important in thetreatment of tumors and other diseases. Anti-angiogenic therapies aretherefore intended for use in the combination treatments of the presentinvention. The combination of a low, relatively frequent dose of atherapeutic agent of the present invention in combination with an agentthat inhibits angiogenesis is particularly contemplated. Exemplaryanti-angiogenic agents that are useful in connection with combinedtherapy are listed above (in connection with immunoconjugates). Any oneor more of such agents, including those in Table B, may be used incombination therapy with the invention. Angiostatin, endostatin,vasculostatin, canstatin and maspin are currently preferred.

[0778] Many known anti-cancer agents also have an anti-angiogenic effectas part of their mechanism of action. These agents, as exemplified bythose in Table E, are particularly contemplated for use in thecombination therapy aspects of the present invention (they may also beconjugated to an antibody of the invention, as described above). TABLE EAnti-Cancer Agents with Anti-Angiogenic Activity Class or Type of AgentExamples Alkylators Cyclophosphamide, edelfosine, estramustine,melphalan Antimetabolites Fluorouracil, methotrexate, mercaptopurine,UFT, tegafur, uracil, cytarabine Anti-Tumor Antibiotics Bleomycin,daunorubicin, doxorubicin, epirubicin, mitomycin, mitoxantroneTopoisomerase Inhibitors Camptothecin, irinotecan, etoposide, topotecanTaxanes Docetaxel, paclitxael Vinca Alkaloids Vinblastine, vincristineMiscellaneous Cisplatin, octreotide

[0779] In addition, the antibody LM609 against the α_(v)β₃ integrin alsoinduces tumor regressions and may be used in combination therapies.Integrin α_(v)β₃ antagonists, such as LM609, induce apoptosis ofangiogenic endothelial cells leaving the quiescent blood vesselsunaffected. LM609 or other α_(v)β₃ antagonists may also work byinhibiting the interaction of α_(v)β₃ and MMP-2, a proteolytic enzymethought to play an important role in migration of endothelial cells andfibroblasts.

[0780] Apoptosis of the angiogenic endothelium by LM609 may have acascade effect on the rest of the vascular network. Inhibiting the tumorvascular network from completely responding to the tumor's signal toexpand may, in fact, initiate the partial or full collapse of thenetwork resulting in tumor cell death and loss of tumor volume. It ispossible that endostatin and angiostatin function in a similar fashion.The fact that LM609 does not affect quiescent vessels but is able tocause tumor regressions suggests strongly that not all blood vessels ina tumor need to be targeted for treatment in order to obtain ananti-tumor effect.

[0781] Antibodies to angiogenin may also be employed, as described inU.S. Pat. No. 5,520,914, specifically incorporated herein by reference.As FGF is connected with angiogenesis, FGF inhibitors may also be used.Certain examples are the compounds having N-acetylglucosaminealternating in sequence with 2-O-sulfated uronic acid as their majorrepeating units, including glycosaminoglycans, such as archaran sulfate.Such compounds are described in U.S. Pat. No. 6,028,061, specificallyincorporated herein by reference, and may be used in combinationherewith.

[0782] N7. VEGF Inhibitors

[0783] VEGF is a multifunctional cytokine that is induced by hypoxia andoncogenic mutations. VEGF is a primary stimulant of the development andmaintenance of a vascular network in embryogenesis. It functions as apotent permeability-inducing agent, an endothelial cell chemotacticagent, an endothelial survival factor, and endothelial cellproliferation factor. Its activity is required for normal embryonicdevelopment, as targeted disruption of one or both alleles of VEGFresults in embryonic lethality.

[0784] The use of one or more VEGF inhibition methods is a preferredaspect of the combination therapies of the present invention. Therecognition of VEGF as a primary stimulus of angiogenesis inpathological conditions has led to various methods to block VEGFactivity. Any of the VEGF inhibitors developed may now be advantageouslyemployed herewith. Accordingly, any one or more of the followingneutralizing anti-VEGF antibodies, soluble receptor constructs,antisense strategies, RNA aptamers and tyrosine kinase inhibitorsdesigned to interfere with VEGF signaling may thus be used.

[0785] Suitable agents include neutralizing antibodies (Kim et al.,1992; Presta et al., 1997; Sioussat et al., 1993; Kondo et al., 1993;Asano et al., 1995), soluble receptor constructs (Kendall and Thomas,1993; Aiello et al., 1995; Lin et al., 1998; Millauer et al., 1996),tyrosine kinase inhibitors (Siemeister et al., 1998), antisensestrategies, RNA aptamers and ribozymes against VEGF or VEGF receptors(Saleh et al., 1996; Cheng et al., 1996). Variants of VEGF withantagonistic properties may also be employed, as described in WO98/16551. Each of the foregoing references are specifically incorporatedherein by reference.

[0786] Blocking antibodies against VEGF will be preferred in certainembodiments, particularly for simplicity. Monoclonal antibodies againstVEGF have been shown to inhibit human tumor xenograft growth and ascitesformation in mice (Kim et al., 1993; Mesiano et al., 1998; Luo et al.,1998a; 1998b; Borgstrom et al., 1996; 1998; each incorporated herein byreference). The antibody A4.6.1 is a high affinity anti-VEGF antibodycapable of blocking VEGF binding to both VEGFR1 and VEGFR2 (Kim et al.,1992; Wiesmann et al., 1997; Muller et al., 1998; Keyt et al., 1996;each incorporated herein by reference). A4.6.1 has recently beenhumanized by monovalent phage display techniques and is currently inPhase I clinical trials as an anti-cancer agent (Brem, 1998; Baca etal., 1997; Presta et al., 1997; each incorporated herein by reference).

[0787] Alanine scanning mutagenesis and X-ray crystallography of VEGFbound by the Fab fragment of A4.6.1 showed that the epitope on VEGF thatA4.6.1 binds is centered around amino acids 89-94. This structural datademonstrates that A4.6.1 competitively inhibits VEGF from binding toVEGFR2, but inhibits VEGF from binding to VEGFR1 most likely by sterichindrance (Muller et al., 1998; Keyt et al., 1996; each incorporatedherein by reference)

[0788] A4.6.1 may be used in combination with the present invention.However, a new antibody termed 2C3 (4545) is currently preferred, whichselectively blocks the interaction of VEGF with only one of the two VEGFreceptors. 2C3 inhibits VEGF-mediated growth of endothelial cells, haspotent anti-tumor activity and selectively blocks the interaction ofVEGF with VEGFR2 (KDR/Flk-1), but not VEGFR1 (FLT-1). In contrast toA4.6.1, 2C3 allows specific inhibition of VEGFR2-induced angiogenesis,without concomitant inhibition of macrophage chemotaxis (mediated byVEGFR1), and is thus contemplated to be a safer therapeutic. U.S. Pat.Nos. 6,342,219, 6,342,221, 6,416,758 and 6,416,758, are specificallyincorporated herein by reference for the purposes of even furtherdescribing the 2C3 antibody and its uses in anti-angiogenic therapy andVEGF inhibition.

[0789] N8. Apoptosis-Inducing Agents

[0790] The therapeutic agents of the present invention are alsopreferably combined with treatment methods that induce apoptosis in anycells within the tumor, including tumor cells and tumor vascularendothelial cells. Exemplary agents that induce apoptosis are listedabove (in connection with immunoconjugates). Any one or more of suchapoptosis-inducing agents may be used in the combination therapies ofthe present invention, without being linked to an antibody of theinvention.

[0791] Many known anti-cancer agents also have an apoptosis-inducingeffect as part of their mechanism of action. These agents, asexemplified by those in Table F, are particularly contemplated for usein the combination therapy aspects of the present invention (they mayalso be conjugated to an antibody of the invention, as described above).TABLE F Anti-Cancer Agents that Induce Apoptosis Class or Type of AgentExamples Antimetabolites Cytarabine, fludarabine, 5-fluoro-29-deoxyuridine, gemcitabine, hydroxyurea, methotrexate DNA Cross-LinkingChlorambucil, cisplatin, cyclophosphamide, Agents nitrogen mustardIntercalating Agents Adriamycin (doxorubicin), mitixantroneTopoisomerase II Poisons Etoposide, teniposide Microtubule-DirectedColcemid, colchicine, docetaxel, vincristine Agents Kinase InhibitorsFlavopiridol, staurosporine, STI571 (CPG 57148B), UCN-01(7-hydroxystaurosporine) Farnesyl Transferase L-739749, L-744832Inhibitors Hormones Glucocorticoids, fenretinide DNA FragmentingBleomycin Agents Hormone Antagonists Tamoxifen, finasteride, LHRHantagonists Biologicals TNF-α, TRAIL, anti-CD20 Protein SynthesisL-asparaginase, cycloheximide, puromycin, Inhibitors diphtheria toxinTopoisomerase II Camptothecin, toptecan Poisons

[0792] N9. Immunotoxins and Coaguligands

[0793] The present invention may also be used in combination with otherimmunotoxins or coaguligands in which the targeting portion is directedto a marker of tumor cells, tumor vasculature or tumor stroma. Any ofthe targeting agents described herein for use in targeting a PE-bindingpeptide to a tumor cell, tumor vasculature or tumor stroma may be usedin these embodiments. In the immunotoxins, the attached agents includeanti-cellular or cytotoxic agents, cytokines, radiotherapeutic agents,anti-angiogenic agents, apoptosis-inducing agents and anti-tubulindrugs. In the coaguligands, the attached agents are coagulants. U.S.Pat. Nos. 5,855,866, 5,965,132, 6,261,535, 6,051,230, 6,451,312(immunotoxins), 6,093,399, 6,004,555, 5,877,289, and 6,036,955(coaguligands) are specifically incorporated herein by reference toexemplify such constructs.

[0794] N10. ADEPT and Prodrug Therapy

[0795] The antibodies of the present invention, including the 9D2, 3G4(ATCC 4545) and like antibodies, may also be used in conjunction withprodrugs, wherein the antibody is operatively associated with aprodrug-activating component, such as a prodrug-activating enzyme, whichconverts a prodrug to the more active form only upon contact with theantibody. This technology is generally termed “ADEPT”, and is describedin, e.g., WO 95/13095; WO 97/26918, WO 97/24143, and U.S. Pat. Nos.4,975,278 and 5,658,568, each specifically incorporated herein byreference.

[0796] The term “prodrug”, as used herein, refers to a precursor orderivative form of a biologically or pharmaceutically active substancethat exerts reduced cytotoxic or otherwise anticellular effects ontargets cells, including tumor vascular endothelial cells, in comparisonto the parent drug upon which it is based. Preferably, the prodrug orprecursor form exerts significantly reduced, or more preferably,negligible, cytotoxic or anticellular effects in comparison to the“native” or parent form. “Prodrugs” are capable of being activated orconverted to yield the more active, parent form of the drug.

[0797] The technical capability to make and use prodrugs exists withinthe skill of the ordinary artisan. Willman et al. (1986) and Stella etal. (1985) are each specifically incorporated herein by reference forpurposes of further supplementing the description and teachingconcerning how to make and use various prodrugs. Exemplary prodrugconstructs that may be used in the context of the present inventioninclude, but are not limited to, phosphate-containing prodrugs (U.S.Pat. No. 4,975,278), thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-based prodrugs (U.S. Pat. Nos.5,660,829; 5,587,161; 5,405,990; WO 97/07118), D-amino acid-modifiedprodrugs, glycosylated prodrugs (U.S. Pat. Nos. 5,561,119; 5,646,298;4,904,768, 5,041,424), β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs (U.S. Pat. No.4,975,278), optionally substituted phenylacetamide-containing prodrugs,and even 5-fluorocytosine (U.S. Pat. No. 4,975,278) and 5-fluorouridineprodrugs and the like, wherein each of the patents are specificallyincorporated herein by reference.

[0798] The type of therapeutic agent or cytotoxic drug that can be usedin prodrug form is virtually limitless. The more cytotoxic agents willbe preferred for such a form of delivery, over, e.g., the delivery ofcoagulants, which are less preferred for use as prodrugs. All that isrequired in forming the prodrug is to design the construct so that theprodrug is substantially inactive and the “released” or activated drughas substantial, or at least sufficient, activity for the intendedpurpose.

[0799] Various improvements on the original prodrugs are also known andcontemplated for use herewith, as disclosed in WO 95/03830; EP 751,144(anthracyclines); WO 97/07097 (cyclopropylindoles); and WO 96/20169. Forexample, prodrugs with reduced Km are described in U.S. Pat. No.5,621,002, specifically incorporated herein by reference, which may beused in the context of the present invention. Prodrug therapy that beconducted intracellularly is also known, as exemplified by WO 96/03151,specifically incorporated herein by reference, and can be practicedherewith.

[0800] For use in ADEPT, the agent that activates or converts theprodrug into the more active drug is operatively attached to an antibodyof the invention. The antibody thus localizes the prodrug convertingcapability within the angiogenic or tumor site, so that active drug isonly produced in such regions and not in circulation or in healthytissues.

[0801] Enzymes that may be attached to the antibodies of the inventionto function in prodrug activation include, but are not limited to,alkaline phosphatase for use in combination with phosphate-containingprodrugs (U.S. Pat. No. 4,975,278); arylsulfatase for use in combinationwith sulfate-containing prodrugs (U.S. Pat. No. 5,270,196); peptidasesand proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidase (U.S. Pat. Nos. 5,660,829; 5,587,161; 5,405,990) andcathepsins (including cathepsin B and L), for use in combination withpeptide-based prodrugs; D-alanylcarboxypeptidases for use in combinationwith D-amino acid-modified prodrugs; carbohydrate-cleaving enzymes suchas β-galactosidase and neuraminidase for use in combination withglycosylated prodrugs (U.S. Pat. Nos. 5,561,119; 5,646,298); β-lactamasefor use in combination with β-lactam-containing prodrugs; penicillinamidases, such as penicillin V amidase (U.S. Pat. No. 4,975,278) orpenicillin G amidase, for use in combination with drugs derivatized attheir amino nitrogens with phenoxyacetamide or phenylacetamide groups;and cytosine deaminase (U.S. Pat. Nos. 5,338,678; 5,545,548) for use incombination with 5-fluorocytosine-based prodrugs (U.S. Pat. No.4,975,278), wherein each of the patents are specifically incorporatedherein by reference.

[0802] Antibodies with enzymatic activity, known as catalytic antibodiesor “abzymes”, can also be employed to convert prodrugs into activedrugs. Abzymes based upon the antibodies of the invention, preferablythe 9D2 and 3G4 and like antibodies, thus form another aspect of thepresent invention. The technical capacity to make abzymes also existswithin those of ordinary skill in the art, as exemplified by Massey etal. (1987), specifically incorporated herein by reference for purposesof supplementing the abzyme teaching. Catalytic antibodies capable ofcatalyzing the breakdown of a prodrug at the carbamate position, such asa nitrogen mustard aryl carbamate, are further contemplated, asdescribed in EP. 745,673, specifically incorporated herein by reference.

[0803] O. Antibody-Coated Liposomes and Therapeutics

[0804] Liposomal formulations are often used in therapeutics andpharmaceuticals. However, the biodistribution of liposomes in initialstudies meant that such formulations were not widely applicable for usein humans. Liposomes are rapidly taken up by the phagocytic cells of thereticuloendothelial system (RES), including the circulating mononuclearphagocytic cells and those located in the liver and spleen. Thus, theblood circulation half-lives can be as short as a few minutes.

[0805] The technology of “stealth or stealthed” liposomes andformulations was thus developed, which allows liposomes to evade uptakeby the RES and circulate for longer (Hristova and Needham, 1993). Apreferred agent for use in stealthing liposomes is polyethylene glycol(PEG), and the resultant liposomes are also termed PEGylated liposomes.Other stealthing agents include poly(2-methyl-2-oxazoline) andpoly(2-ethyl-2-oxazoline) conjugates (Woodle et al., 1994). A range ofimproved stealthed liposomes are described in U.S. Pat. No. 6,284,267,specifically incorporated herein by reference, which may be used incombination with the present invention.

[0806] Liposomes smaller in diameter than the average diameter of thefenestrae in capillaries leak out from the circulation. The averagediameter of the fenestrae in rapidly growing tumors is larger than innormal tissues and therefore liposomes smaller than about 100 nm indiameter migrate into tumors. Stealth liposomes have thus been proposedfor use in delivering cytotoxic agents to tumors in cancer patients. Arange of drugs have been incorporated into stealth liposomes, includingcisplatin (Rosenthal et al., 2002), TNFα (Kim et al., 2002), doxorubicin(Symon et al., 1999) and adriamycin (Singh et al., 1999), each referencebeing specifically incorporated herein by reference. However, recentreports have indicated unexpected low efficacy of stealth liposomaldoxorubicin and vinorelbine in the treatment of metastatic breast cancer(Rimassa et al., 2003).

[0807] The present invention provides improved stealthed liposomeformulations, overcoming various of the drawbacks in the art, in whichthe stealthed liposomes are functionally associated or “coated” with anantibody that binds to an aminophospholipid or anionic phospholipid,preferably to PS or PE. The 9D2, 3G4 (ATCC 4545) and like, competingantibodies of the invention are preferred for such uses, although anyantibody, or antigen binding region thereof, which binds to anaminophospholipid or anionic phospholipid may be used. A divalentantibody or antibody portion is not required in these aspects of theinvention.

[0808] Any stealthed liposome may form the basis of the new liposomalformulations, and preferably a PEGylated liposome will be employed. Thestealthed liposomes are “coated”, i.e., operatively or functionallyassociated with the antibody that binds to an aminophospholipid oranionic phospholipid. The operative or functional association is madesuch that the antibody retains the ability to specifically bind to thetarget aminophospholipid or anionic phospholipid, preferably PS or PE,thereby delivering or targeting the stealthed liposome and any contentsthereof to PS- and/or PE-positive cells, such as tumor cells and tumorvascular endothelial cells.

[0809] The antibody-coated stealthed liposomes of the invention may beused alone. Preferably, however, such liposomes will also contain one ormore second therapeutic agents, such as anti-cancer or chemotherapeuticagents (the first therapeutic agent being the antibody itself). Thesecond therapeutic agents are generally described as being within the“core” of the liposome. Any one or more of the second, anti-cancer orchemotherapeutic agents known in the art and/or described herein forconjugation to antibodies, or for combination therapies, may be used inthe antibody-coated stealthed liposomes of the invention. For example,any chemotherapeutic or radiotherapeutic agent, cytokine,anti-angiogenic agent or apoptosis-inducing agent. Currently preferredwithin the chemotherapeutic agents are anti-tubulin drugs, docetaxel andpaclitaxel.

[0810] Moreover, the antibody-coated stealthed liposomes of theinvention may also be loaded with one or more anti-viral drugs for usein treating viral infections and diseases. As with the anti-canceragents, any one or more of the second, anti-viral drugs known in the artand/or described herein for conjugation to antibodies, or forcombination therapies, may be used in the antibody-coated stealthedliposomes of the invention. Cidofovir and AZT are currently preferredexamples.

[0811] P. Anti-Vascular, Anti-Angiogenic and Other Therapies

[0812] The present invention may also be used in the treatment of otherdiseases in which aberrant vasculature is involved, including diseasesand disorders having prothrombotic blood vessels. Although not the onlytherapeutic mechanism, the antibodies, immunoconjugates andpeptide-based therapeutics of the present invention may also be used totreat animals and patients with aberrant angiogenesis, such as thatcontributing to a variety of diseases and disorders.

[0813] Whether based upon anti-angiogenesis, prothrombotic vasculatureor other anti-vascular mechanisms, the present invention may thus beused to treat prevalent and/or clinically important diseases outside thefield of cancer, including arthritis, rheumatoid arthritis, psoriasis,atherosclerosis, diabetic retinopathy, age-related macular degeneration,Grave's disease, vascular restenosis, including restenosis followingangioplasty, arteriovenous malformations (AVM), meningioma, hemangiomaand neovascular glaucoma. Other targets for intervention includeangiofibroma, atherosclerotic plaques, corneal graft neovascularization,hemophilic joints, hypertrophic scars, osler-weber syndrome, pyogenicgranuloma retrolental fibroplasia, scleroderma, trachoma, vascularadhesions, synovitis, dermatitis, various other inflammatory diseasesand disorders, and even endometriosis. Further diseases and disordersthat are treatable by the invention, and the unifying basis of suchdisorders, are set forth below.

[0814] One prominent disease in which aberrant vasculature andangiogenesis is involved is rheumatoid arthritis, wherein the bloodvessels in the synovial lining of the joints undergo angiogenesis. Inaddition to forming new vascular networks, the endothelial cells releasefactors and reactive oxygen species that lead to pannus growth andcartilage destruction. The factors involved in angiogenesis may activelycontribute to, and help maintain, the chronically inflamed state ofrheumatoid arthritis. Factors associated with angiogenesis also have arole in osteoarthritis, contributing to the destruction of the joint.Various factors, including VEGF, have been shown to be involved in thepathogenesis of rheumatoid arthritis and osteoarthritis.

[0815] Another important example of a disease involving aberrantvasculature and angiogenesis is ocular neovascular disease. This diseaseis characterized by invasion of new blood vessels into the structures ofthe eye, such as the retina or cornea. It is the most common cause ofblindness and is involved in approximately twenty eye diseases. Inage-related macular degeneration, the associated visual problems arecaused by an ingrowth of chorioidal capillaries through defects inBruch's membrane with proliferation of fibrovascular tissue beneath theretinal pigment epithelium. Angiogenic damage is also associated withdiabetic retinopathy, retinopathy of prematurity, corneal graftrejection, neovascular glaucoma and retrolental fibroplasia.

[0816] Other diseases associated with corneal neovascularization thatcan be treated according to the present invention include, but are notlimited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contactlens overwear, atopic keratitis, superior limbic keratitis, pterygiumkeratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis,Mycobacteria infections, lipid degeneration, chemical burns, bacterialulcers, fungal ulcers, Herpes simplex infections, Herpes zosterinfections, protozoan infections, Kaposi sarcoma, Mooren ulcer,Terrien's marginal degeneration, mariginal keratolysis, rheumatoidarthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis,Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, andcorneal graph rejection.

[0817] Diseases associated with retinal/choroidal neovascularizationthat can be treated according to the present invention include, but arenot limited to, diabetic retinopathy, macular degeneration, sickle cellanemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease,vein occlusion, artery occlusion, carotid obstructive disease, chronicuveitis/vitritis, mycobacterial infections, Lyme's disease, systemiclupus erythematosis, retinopathy of prematurity, Eales disease, Bechetsdisease, infections causing a retinitis or choroiditis, presumed ocularhistoplasmosis, Bests disease, myopia, optic pits, Stargarts disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, trauma and post-laser complications.

[0818] Other diseases that can be treated according to the presentinvention include, but are not limited to, diseases associated withrubeosis (neovascularization of the angle) and diseases caused by theabnormal proliferation of fibrovascular or fibrous tissue including allforms of proliferative vitreoretinopathy, whether or not associated withdiabetes.

[0819] Chronic inflammation also involves aberrant vasculature andpathological angiogenesis. Such disease states as ulcerative colitis andCrohn's disease show histological changes with the ingrowth of new bloodvessels into the inflamed tissues. Bartonellosis, a bacterial infectionfound in South America, can result in a chronic stage that ischaracterized by proliferation of vascular endothelial cells.

[0820] Another pathological role associated with aberrant vasculatureand angiogenesis is found in atherosclerosis. The plaques formed withinthe lumen of blood vessels have been shown to have angiogenicstimulatory activity. There is particular evidence of thepathophysiological significance of angiogenic markers, such as VEGF, inthe progression of human coronary atherosclerosis, as well as inrecanalization processes in obstructive coronary diseases. The presentinvention provides an effective treatment for such conditions.

[0821] One of the most frequent angiogenic diseases of childhood is thehemangioma. In most cases, the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, the hemangiomatoses, have a highmortality rate. Therapy-resistant hemangiomas exist that cannot betreated with therapeutics currently in use, but are addressed by theinvention.

[0822] Angiogenesis is also responsible for damage found in hereditarydiseases such as Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia. This is an inherited disease characterized by multiplesmall angiomas, tumors of blood or lymph vessels. The angiomas are foundin the skin and mucous membranes, often accompanied by epistaxis(nosebleeds) or gastrointestinal bleeding and sometimes with pulmonaryor hepatic arteriovenous fistula.

[0823] Angiogenesis is also involved in normal physiological processessuch as reproduction and wound healing. Angiogenesis is an importantstep in ovulation and also in implantation of the blastula afterfertilization. Prevention of angiogenesis according to the presentinvention could be used to induce amenorrhea, to block ovulation or toprevent implantation by the blastula. In wound healing, excessive repairor fibroplasia can be a detrimental side effect of surgical proceduresand may be caused or exacerbated by angiogenesis. Adhesions are afrequent complication of surgery and lead to problems such as smallbowel obstruction. This can also be treated by the invention.

[0824] Each of the foregoing diseases and disorders, along with alltypes of tumors, are also contemplated for treatment according to thepresent invention. U.S. Pat. No. 5,712,291 is specifically incorporatedherein by reference to further demonstrate the knowledge in the art thatonce the inhibition of angiogenesis has been shown using a particularagent, the treatment of an extensive range of diseases associated withaberrant angiogenesis using that and like agents can reasonably becarried out. U.S. Pat. No. 6,524,583 is also specifically incorporatedherein by reference for similar purposes and to particularly demonstratethat this principle applies to the inhibition of angiogenesis and thetreatment of angiogenic diseases using antibody-based therapeutics. Theanti-angiogenic effects of the 3G4 antibody (ATCC 4545) in tumor-bearingmice (FIG. 17A) is thus important evidence that 3G4 and like antibodiesare suitable for treating a wide range of angiogenic diseases.

[0825] The invention further provides compositions and methods for usein treating other diseases in which aminophospholipids and/or anionicphospholipids, particularly PS and PE, play a role. For example, as PSis involved in cell adhesion, inflammatory responses and septic shock,antibodies to PS can be used in the treatment of inflammation and septicshock. The use of the 3G4 (ATCC 4545) or like antibodies is preferredfor such embodiments, particularly an Fab dimer of such an antibody. Aduramycin Fab dimer is also particularly contemplated for use intreating septic shock.

[0826] Aminophospholipids and/or anionic phospholipids, particularly PS,are also involved in sickle cell anaemia, in particular, as part of theclearance mechanism. Antibodies to PS can therefore be used to treat orameliorate sickle cell anaemia. The use of the 3G4 (ATCC 4545) or likeantibodies is preferred, particularly an Fab dimer thereof.

[0827] Most bacteria express the anionic phospholipid, PA. Antibodiesthat bind to PA, optionally with binding to other anionic phospholipids,can therefore be used as anti-bacterial agents. Although the antibodiesof the invention can be prepared in E. coli, and are thus notbacteriocidal in all circumstances, an anti-bacterial role in vivo isbelieved to result from the ability to fix complement. An intactantibody rather than an antibody fragment should therefore be used as ananti-bacterial agent. The 3G4 (ATCC 4545) and like antibodies arepreferred for use in such embodiments, although any antibody that fixescomplement and binds to PA may be employed, such as other PA-bindingantibodies from Table 4.

[0828] Antiphospholipid syndrome and lupus, autoimmune disorders inwhich antibodies are produced against the body's own phospholipids, areassociated with coagulation disorders, including miscarriages andthrombocytopenia (low platelet counts). Accordingly, theanti-phospholipid antibodies in these patients are pathogenicantibodies, which cause thrombosis. The antibodies of the presentinvention, however, bind to aminophospholipids and anionic phospholipidswithout exhibiting such side effects. Accordingly, the antibodies of theinvention are contemplated for use in treating antiphospholipidsyndrome, associated diseases and complications thereof.

[0829] The pathogenic anti-phospholipid antibodies that circulate inpatients with antiphospholipid syndrome are believed to bind to PS, PEand other phospholipids in combination with proteins, such asβ₂-glycoprotein I, prothrombin, kininogens, prekallikrein and factor XI(Rote, 1996; Sugi and McIntyre, 1995; 1996a; 1996b). β₂-glycoprotein Iand prothrombin bound to PS are reported to be the primary antigens foranti-cardiolipin antibodies and lupus antibodies, respectively. Theantibodies of the present invention have been particularly selected onthe basis of not binding to aminophospholipids and anionic phospholipidsonly in the presence of serum proteins. Therefore, by binding to thephospholipid component, the antibodies of the invention are contemplatedfor use in antagonizing or competing with the pathogenic antibodies insuch patients, thus displacing the pathogenic antibodies from theirphospholipid-protein targets in the body.

[0830] Q. PE-Binding Peptide Derivatives and Conjugates

[0831] In addition to antibodies and immunoconjugates, the presentinvention further provides PE-binding peptide derivatives and varioususes, particularly in the treatment of tumors and viral diseases.Currently preferred PE-binding peptide constructs and derivatives arethose based upon the peptide termed duramycin. Three general categoriesof PE-binding peptide and duramycin derivatives are provided by theinvention, two of which use the PE-binding peptide or duramycin as thetargeting portion of the construct, and the other uses the duramycin orlike agent mainly as the effector portion of the construct.

[0832] The use of PE-binding peptides, preferably duramycin, astargeting agents is based on their ability to impart a selective bindingcapacity to a resultant construct. Accordingly, a construct or conjugatecontaining a PE-binding peptide, preferably duramycin, will specificallybind to PE-expressing cells, such as tumor vascular endothelial cells,malignant tumor cells, proliferating cells and/or virally infectedcells.

[0833] As PE-binding peptides such as duramycin have biological activityin addition to the PE targeting function, it is not necessary toconjugate a PE-binding peptide such as duramycin to a therapeutic agentto achieve a therapeutic conjugate. However, as PE-binding peptides suchas duramycin have associated toxicities in their natural form, thepeptide should be modified to reduce toxicity. The toxicities areconnected with the ability of the peptides to form clusters, form poresin cell membranes, and to generally permeate or penetrate into thecells. Accordingly, these functions should be attenuated, tosignificantly or substantially prevent the PE-binding peptide fromforming clusters, permeating into the cells and being nonspecificallytoxic. Preferably, whilst the ability to bind to PE is substantiallymaintained, the ability of the PE-binding peptides to form clusters andpenetrate cells is substantially inhibited, thus significantly reducingor abolishing cytotoxicity.

[0834] The first category of PE-binding peptide derivatives with reducedtoxicity provided by the present invention is that in which thePE-binding peptide, preferably duramycin, is rendered relatively orsubstantially cell impermeant. This is preferably achieved by attachingto a cell impermeant group, which can be a small group with positive ornegative charge or a polar group, or can be in the form of an inertcarrier. The terms “cell impermeant group” and “cell impermeantPE-binding peptide”, as used herein, are relative rather than absolute,and refer to modified PE-binding peptides, preferably duramycin, inwhich the ability to form clusters and permeate cells has beensignificantly, and preferably substantially, reduced. The resultant cellimpermeant PE-binding peptides may function by trapping PE, andassociated membrane molecules, on the exterior of cells and/or bybringing host defenses to bear on the peptide-coated cells.

[0835] Within this category of PE-binding peptide derivatives, certainconstructs will emphasize the recruitment of host defenses, thusenhancing their therapeutic activity. For example, where a PE-bindingpeptide, preferably duramycin, is attached to an immunoglobulin, theimmunoglobulin can function both as an inert carrier and as an immuneeffector. This applies to immunoglobulins of so-called “irrelevantspecificity” and to immunoglobulin derivatives without antigen bindingcapacity, such as Fc regions. By virtue of the attached immunoglobulinor immunoglobulin derivative, such constructs will be able to redirecthost defenses against PE-expressing cells, e.g. by attracting and/oractivating immune effector cells.

[0836] In the second general category of PE-binding peptide derivativesof the invention, the peptides are still modified to reduce cellpenetration and resultant toxicity, but rather than using a small cellimpermeant group or inert carrier, an agent is used that changes theblood and tissue distribution of the resultant construct. Preferredexamples are those in which a PE-binding peptide, preferably duramycin,is attached to a targeting agent that binds to a component of a tumorcell, tumor or intratumoral vasculature or tumor stroma. Although thePE-binding peptide itself still has a targeting property, in theseaspects of the invention, the targeting agent primarily directs theconstruct to the target tissue, such as to the tumor environment, andthe attached PE-binding peptide such as duramycin exerts a therapeuticeffect upon delivery.

[0837] The third general category of PE-binding peptide derivativesreturns to the use of the PE-binding peptide, preferably duramycin, as atargeting agent to localize the derivative to PE-expressing cells. Asvirally infected cells express PE at the cell surface, as opposed tonormal, uninfected cells, linking a PE-binding peptide such as duramycinto an anti-viral agent will provide an effective, targeted anti-viralagent. Although the PE-binding peptide portion, preferably duramycin,may have additional therapeutic effects, the attached anti-viral agentis designed to be the primary therapeutic agent within such constructs.

[0838] Any of the conjugation techniques described above may be used toprepare duramycin derivatives in accordance with the invention,including cross-linkers, peptide spacers, biotin:avidin constructs andrecombinant expression. An advantageous site of attachment within theduramycin molecule, for example, is to the lysine residue at amino acidposition 2 in the duramycin sequence (SEQ ID NO:9; FIG. 13P; Hayashi etal., 1990). However, linkage at this site is not a requirement of theinvention.

[0839] Accordingly, PE-binding peptides, preferably duramycin, can bederivatized to have a functional group available for cross-linkingpurposes. A wide variety of groups can be used in this manner, forexample, primary or secondary amine groups, hydrazide or hydrazinegroups, carboxyl alcohol, phosphate, carbamate and alkylating groups.The agents for attachment, including anti-viral agents, may thus beconjugated through a Schiffs base linkage, a hydrazone or acyl hydrazonebond or a hydrazide linker (U.S. Pat. Nos. 5,474,765 and 5,762,918, eachspecifically incorporated herein by reference).

[0840] Q1. PE-Binding and Anti-Microbial Peptides

[0841] Any PE-binding peptide may be used in these aspects of theinvention. For example, low and high molecular weight kininogens areknown to bind PE. The protein and DNA sequences for a variety of suchbinding proteins, including the human proteins, are known in the art,facilitating the use of PE-binding peptides therefrom. For example, thehuman genes and proteins for high and low molecular weight kininogensare described in Kitamura et al. (1985) and Kellermann et al. (1986),each specifically incorporated herein by reference.

[0842] U.S. Pat. No. 6,312,694 describes certain PE-binding conjugatesusing PE-binding proteins, such as kininogens, and PE-binding fragmentsthereof. In U.S. Pat. No. 6,312,694, the PE-binding proteins orPE-binding fragments thereof are operatively attached to anti-cellularagents, toxins and coagulation factors. In the present case, PE-bindingpeptides are attached to inert carriers, tumor targeting agents oranti-viral agents. Although the present agents for attachment and theirmethods of use represent surprising advances, U.S. Pat. No. 6,312,694 isspecifically incorporated herein by reference for purposes of furtherdescribing and enabling PE-binding peptides, such as PE-binding peptidefragments of kininogens.

[0843] Currently preferred PE-binding peptides for use in the inventionare those based upon the PE-binding molecule, duramycin. Duramycin(2622U90, Moli1901) is an antimicrobial peptide from the lantibioticfamily (U.S. Pat. No. 4,452,782; Shotwell et al., 1958; Nakamura andRacker, 1984), and other members of the lantibiotic family may be usedin the present invention. Where the PE-binding peptides are used as thetargeting agent of the construct, for example, when linked to an inertcarrier or to an anti-viral agent, a lantibiotic PE-binding peptideshould substantially retain PE binding activity. When used as thetherapeutic agent in a construct, particularly when attached to atumor-targeting agent, there is more tolerance for some loss of PEbinding activity.

[0844] Testing a candidate peptide to confirm or identify those thatsubstantially bind to PE is a straightforward matter in light of thepresent disclosure and can be achieved, for example, using any one ormore of the ELISAs described herein. Lantibiotics for use as PE-bindingpeptides herein will preferably exhibit substantially the same PEbinding activity as duramycin, and even more preferably, will alsoexhibit substantially the same specificity for PE over otherphospholipids as duramycin. Such properties can also be readilydetermined in light of the present disclosure, particularly the workingexamples.

[0845] Based upon the criteria above, the following lantibiotics may beused as part of the constructs and conjugates of the present invention:duramycin, cinnamycin, actagardine, ancovenin, epidermin, gallidermin,lanthiopeptin, mersacidin, nisin, Pep5 and subtilin. Duramycin is themost preferred PE-binding peptide for use in all aspects of theinvention. Duramycin is an antimicrobial, which has also been suggestedfor use in treating asthma, chronic bronchitis and Mycobacteriumtuberculosis infection (U.S. Pat. Nos. 5,849,706; 5,716,931; 5,683,675;5,651,957; and 5,512,269; each specifically incorporated herein byreference) and cystic fibrosis (McNulty et al., 2003). However,duramycin has not previously been described or suggested for conjugationto a cell impermeant group, particularly not for use in treating viralinfections.

[0846] Cinnamycin (Ro09-0198) is a related molecule that binds to PE(Wakamatsu et al., 1986; Choung et al., 1988a; 1988b). Labeledcinnamycin has been used as a probe to study the transbilayer movementof PE (Aoki et al., 1994; Emoto et al., 1996) and PE exposure duringapoptosis of T cells in vitro (Emoto et al., 1997; Umeda and Emoto,1999). However, therapeutic uses of cinnamycin derivatives in accordancewith the present invention have not been previously described orsuggested. Pharmaceutical compositions containing PE-binding peptidederivatives of the invention based upon cinnamycin, and various medicaluses thereof, therefore represent an advance in the art, particularlywhere such compositions are intended for use in treating viralinfections.

[0847] The following anti-microbial peptides may also be used in theconjugates of the invention, particularly as therapeutic agents attachedto tumor targeting agents: cystibiotics, such as pediocin AcH/PA1,leucocin A/Ual 187, mesentericin Y 105, sakacin A, sakacin P, lactacinF, cerein 7/8 and carnobacteriocins, such as carnobacteriocin A, BM1 andB2; and thiolbiotics, particularly lactococcins, such as lactococcin B,A, M^(a), N^(a), G^(a) and G.

[0848] Q2. Cell Impermeant Groups

[0849] Attaching a PE-binding peptide, preferably duramycin, to a cellimpermeant group will reduce the ability of the peptides to formclusters, substantially preventing the PE-binding peptide frompermeating into normal cells and thus reducing the toxicity. The PEbinding property is maintained, however, so that the peptides canlocalize to aberrant or infected cells, which have PE exposed on thesurface.

[0850] Exemplary cell impermeant groups include groups that bearpositive or negative charge at physiological pH, such as sulfate,sulfonate, phosphate, carboxyl, phenolic, quaternary ammonium ions andamine groups. Further examples are polar groups, such as simple sugarsand polysaccharides, amino acids and polyalcohols. Duramycin, inparticular, may be linked to biotin to form biotinylated PE-bindingpeptides, which can be dispersed in a pharmaceutical composition ormedicament, particularly one intended for treating a viral infection.The cell impermeant group can also be a polypeptide, protein orimmunoglobulin, any of can function as an inert carrier or as atargeting agent.

[0851] Q3. Inert Carriers

[0852] PE-binding peptides, preferably duramycin, can be rendered cellimpermeant by attachment to an inert, cell impermeant carrier. A widerange of inert, cell impermeant carriers can be conjugated to aPE-binding peptide, preferably duramycin, to prepare a cell impermeantPE-binding peptide, so long as PE binding activity is not substantiallydestroyed. The inert carriers should preferably be biologicallycompatible, such that they do not result in any significant untowardeffects upon administration to an animal or patient.

[0853] Carrier proteins can be used, and exemplary proteins are albuminsand globulins. Neutravidin and streptavidin will often be preferred.Non-protein carriers can also be used, such as natural or syntheticpolymers, including polysaccharides and PEG.

[0854] In certain embodiments, the carrier will be an immunoglobulin orportion thereof. Human immunoglobulins (HIgG) will be preferred forhuman administration. Immunoglobulins can also impart targetingfunctions, as discussed below. As an inert carrier, an immunoglobulin isone of “irrelevant specificity”, in that it does not impart a targetingfunction to the conjugate. However, certain advantages may still beachieved through the selection of particular types of immunoglobulin.For example, the Fc portion of an immunoglobulin may be used to recruithost immune cells and thus further stimulate host defenses.

[0855] Q4. Targeting Agents

[0856] Rather than attaching to an inert carrier, PE-binding peptides,preferably duramycin, can be rendered cell impermeant by attachment to atargeting agent, in particular, one that binds to a component of a tumorcell, tumor or intratumoral vasculature or tumor stroma. The targetingagent directs the construct to the target tissue, preferably the tumorenvironment, and the attached PE-binding peptide, preferably duramycin,exerts a therapeutic effect upon delivery.

[0857] Suitable targeting agents are components, such as antibodies andother agents, which bind to a tumor cell. Agents that “bind to a tumorcell” are defined herein as targeting agents that bind to any accessiblecomponent or components of a tumor cell, or that bind to a componentthat is itself bound to, or otherwise associated with, a tumor cell, asfurther described herein.

[0858] The majority of such tumor cell-targeting agents and bindingligands are contemplated to be agents, particularly antibodies, thatbind to a cell surface tumor antigen or marker. Many such antigens areknown, as are a variety of antibodies for use in antigen binding andtumor targeting. The invention thus includes targeting agents that bindto an identified tumor cell surface antigen and/or that bind to anintact tumor cell. The identified tumor cell surface antigens and intacttumor cells of Table I and Table II of U.S. Pat. Nos. 5,877,289;6,004,555; 6,036,955; 6,093,399 are specifically incorporated herein byreference for the purpose of exemplifying suitable tumor cell surfaceantigens.

[0859] Examples of tumor cell binding regions are those that comprise anantigen binding region of an antibody that binds to the cell surfacetumor antigen p185^(HER2), milk mucin core protein, TAG-72, Lewis a orcarcinoembryonic antigen (CEA). Another group of tumor cell bindingregions are those that comprise an antigen binding region of an antibodythat binds to a tumor-associated antigen that binds to the antibody9.2.27, OV-TL3, MOv18, B3 (ATCC HB 10573), KS1/4 (obtained from a cellcomprising the vector pGKC2310 (NRRL B-18356) or the vector pG2A52 (NRRLB-18357), 260F9 (ATCC HB 8488) or D612 (ATCC HB 9796). D612 is describedin U.S. Pat. No. 5,183,756, and has ATCC Accession No. HB 9796; B3 isdescribed in U.S. Pat. No. 5,242,813, and has ATCC Accession No. HB10573; and recombinant and chimeric KS1/4 antibodies are described inU.S. Pat. No. 4,975,369; each incorporated herein by reference.

[0860] Targetable components of tumor cells further include componentsreleased from necrotic or otherwise damaged tumor cells, includingcytosolic and/or nuclear tumor cell antigens. These are preferablyinsoluble intracellular antigen(s) present in cells that may be inducedto be permeable, or in cell ghosts of substantially all neoplastic andnormal cells, that are not present or accessible on the exterior ofnormal living cells of a mammal.

[0861] U.S. Pat. Nos. 5,019,368, 4,861,581 and 5,882,626, issued to AlanEpstein and colleagues, are each specifically incorporated herein byreference for purposes of even further describing and teaching how tomake and use antibodies specific for intracellular antigens that becomeaccessible from malignant cells in vivo. The antibodies described aresufficiently specific to internal cellular components of mammalianmalignant cells, but not to external cellular components. Exemplarytargets include histones, but all intracellular components specificallyreleased from necrotic tumor cells are encompassed.

[0862] Upon administration to an animal or patient with a vascularizedtumor, such antibodies localize to the malignant cells by virtue of thefact that vascularized tumors naturally contain necrotic tumor cells,due to the process(es) of tumor re-modeling that occur in vivo and causeat least a proportion of malignant cells to become necrotic. Inaddition, the use of such antibodies in combination with other therapiesthat enhance tumor necrosis serves to enhance the effectiveness oftargeting and subsequent therapy. These types of antibodies may thus beused as targeting agents as disclosed herein.

[0863] A range of suitable targeting agents are available that bind tomarkers present on tumor endothelium and stroma, but largely absent fromnormal cells, endothelium and stroma. For tumor vasculature targeting,the targeting antibody or ligand will often bind to a marker expressedby, adsorbed to, induced on or otherwise localized to the intratumoralblood vessels of a vascularized tumor. “Components of tumor vasculature”thus include both tumor vasculature endothelial cell surface moleculesand any components, such as growth factors, that may be bound to thesecell surface receptors or molecules. The following patents arespecifically incorporated herein by reference for the purposes of evenfurther supplementing the present teachings regarding the preparationand use of targeting agents directed against expressed, adsorbed,induced or localized markers of tumor vasculature: U.S. Pat. Nos.5,855,866; 5,776,427; 5,863,538; 5,660,827; 5,855,866; 5,877,289;6,004,554; 5,965,132; 6,036,955; 6,093,399; 6,004,555.

[0864] Examples of surface-expressed targets of tumor and intratumoralblood vessels include vascular cell surface receptors and cell adhesionmolecules (Thorpe and Ran, 2000, specifically incorporated herein byreference, see Table 1). Suitable examples include endoglin, targetedby, e.g., TEC-4, TEC-11, E-9 and Snef antibodies; E-selectin, targetedby, e.g., H4/18 antibodies; VCAM-1, targeted by, e.g., E1/6 and 1.4c3antibodies; endosialin, targeted by, e.g., FB5 antibodies; α_(v)β₃integrin, targeted by, e.g., LM609 and peptide targeting agents; theVEGF receptor VEGFR1, targeted by a number of antibodies, andparticularly by VEGF; the VEGF receptor complex, also targeted by anumber of antibodies, such as 3E7 and GV39; and PSMA, targeted byantibodies such as J591. Examples such as endoglin, TGFβ receptors,E-selectin, P-selectin, VCAM-1, ICAM-1, a ligand reactive with LAM-1, aVEGF/VPF receptor, an FGF receptor, α_(v)β₃ integrin, pleiotropin,endosialin are further described and enabled in U.S. Pat. Nos.5,855,866; 5,877,289; 6,004,555; 6,093,399; each incorporated herein byreference.

[0865] Further suitable examples include proteoglycans, such as NG2, andmatrix metalloproteinases (MMPs), such as MMP2 and MMP9, each targetedby particular peptide targeting agents (Thorpe and Ran, 2000). These areexamples of remodeling enzymes that are expressed as targetable entitiesin the tumor, which is a site of vascular remodeling. Further suitabletargets are thrombomodulin, Thy-1 and cystatin. Studies identifyingsequences elevated in tumor endothelium have also identifiedthrombomodulin, MMP 11 (stromelysin), MMP 2 (gelatinase) and variouscollagens as targetable tumor vascular markers, which is also inaccordance with U.S. Pat. Nos. 6,004,555 and 6,093,399, specificallyincorporated herein by reference.

[0866] Another suitable target is PSMA (prostate-specific membraneantigen). PSMA, initially defined by monoclonal antibody 7E11, wasoriginally identified as a marker of prostate cancer and is known to bea type 2 integral membrane glycoprotein. The 7E11 antibody binds to anintracellular epitope of PSMA that, in viable cells, is not availablefor binding. In the context of the present invention, PSMA is thustargeted using antibodies to the extracellular domain. Such antibodiesreact with tumor vascular endothelium in a variety of carcinomas,including lung, colon and breast, but not with normal vascularendothelium.

[0867] Many antibodies that bind to the external domain of PSMA arereadily available and may be used in the present invention. Monoclonalantibodies 3E11, 3C2, 4E10-1.14, 3C9 and 1G3 display specificities fordiffering regions of the extracellular domain of the PSMA protein andare suitable for use herein. Three additional antibodies to theextracellular domain of PSMA are J591, J415 and PEQ226.5, which confirmPSMA expression in tumor-associated vasculature and may be used in theinvention. As the nucleic acids encoding PSMA and variants thereof arealso readily available, U.S. Pat. Nos. 5,935,818 and 5,538,866,additional antibodies can be generated if desired.

[0868] U.S. Pat. No. 6,150,508, specifically incorporated herein byreference, describes various other monoclonal antibodies that bind tothe extracellular domain of PSMA, which may be used in the presentinvention. Any one or more of the thirty-five exemplary monoclonalantibodies reactive with PSMA expressed on the cell surface may be used.These include, 3F5.4G6 (ATCC HB12060); 3D7-1.I. (ATCC HB12309);4E10-1.14 (ATCC HB12310); 3E11 (ATCC HB12488); 4D8 (ATCC HB12487); 3E6(ATCC HB12486); 3C9 (ATCC HB12484); 2C7 (ATCC HB12490); 1G3 (ATCCHB12489); 3C4 (ATCC HB12494); 3C6 (ATCC HB12491); 4D4 (ATCC HB12493);1G9 (ATCC HB12495); 5C8B9 (ATCC HB12492); 3G6 (ATCC HB12485); and 4C8B9(ATCC HB12492).

[0869] Further antibodies, or binding portions thereof, that recognizean extracellular domain of PSMA are described in U.S. Pat. Nos.6,107,090 and 6,136,311, each specifically incorporated herein byreference. Four hybridoma cell lines in particular are described, beingE99, J415, J533, and J591 (ATCC HB-12101, HB-12109, HB-12127, andHB-12126), any one or more of which may thus be used as a targetingagent in accordance with the claimed invention.

[0870] Targeting agents that bind to “adsorbed” targets are anothersuitable group, such as those that bind to ligands or growth factorsthat bind to tumor or intratumoral vasculature cell surface receptors.Such antibodies include those that bind to VEGF, FGF, TGFβ, HGF, PF4,PDGF, TIMP or a tumor-associated fibronectin isoform (U.S. Pat. Nos.5,877,289; 5,965,132; 6,093,399 and 6,004,555; each incorporated hereinby reference).

[0871] Other suitable targeting antibodies, or fragments thereof, arethose that bind to epitopes that are present on ligand-receptorcomplexes or growth factor-receptor complexes, but absent from both theindividual ligand or growth factor and the receptor. Such antibodieswill recognize and bind to a ligand-receptor or growth factor-receptorcomplex, as presented at the cell surface, but will not bind to the freeligand or growth factor or the uncomplexed receptor. A “bound receptorcomplex”, as used herein, therefore refers to the resultant complexproduced when a ligand or growth factor specifically binds to itsreceptor, such as a growth factor receptor.

[0872] These aspects are exemplified by the VEGF/VEGF receptor complex.Such ligand-receptor complexes will be present in a significantly highernumber on tumor-associated endothelial cells than on non-tumorassociated endothelial cells, and may thus be targeted by anti-complexantibodies. Anti-complex antibodies include the monoclonal antibodies2E5, 3E5 and 4E5 and fragments thereof.

[0873] Antigens naturally and artificially inducible by cytokines andcoagulants may also be targeted. Exemplary cytokine-inducible antigensare E-selectin, VCAM-1, ICAM-1, endoglin, a ligand reactive with LAM-1,and even MHC Class II antigens, which are induced by, e.g., IL-1, IL-4,TNF-α, TNF-β or IFN-γ, as may be released by monocytes, macrophages,mast cells, helper T cells, CD8-positive T-cells, NK cells or even tumorcells.

[0874] Further inducible antigens include those inducible by acoagulant, such as by thrombin, Factor IX/IXa, Factor X/Xa, plasmin or ametalloproteinase (matrix metalloproteinase, MMP). Generally, antigensinducible by thrombin will be used. This group of antigens includesP-selectin, E-selectin, PDGF and ICAM-1, with the induction andtargeting of P-selectin and/or E-selectin being generally preferred.

[0875] In other embodiments, the vasculature and stroma targeting agents(see below) of the invention will be targeting agents that arethemselves biological ligands, or portions thereof, rather than anantibodies. “Biological ligands” in this sense will be those moleculesthat bind to or associate with cell surface molecules, such asreceptors, that are accessible in the stroma or on vascular cells; asexemplified by cytokines, hormones, growth factors, and the like. Anysuch growth factor or ligand may be used so long as it binds to thedisease-associated stroma or vasculature, e.g., to a specific biologicalreceptor present on the surface of a tumor vasculature endothelial cell.

[0876] Suitable growth factors for use in these aspects of the inventioninclude, for example, VEGF/VPF (vascular endothelial growthfactor/vascular permeability factor), FGF (the fibroblast growth factorfamily of proteins), TGFβ (transforming growth factor B), atumor-associated fibronectin isoform, scatter factor/hepatocyte growthfactor (HGF), platelet factor 4 (PF4), PDGF (platelet derived growthfactor), TIMP or even IL-8, IL-6 or Factor XIIIa. VEGF/VPF and FGF willoften be preferred.

[0877] Further suitable targeting agents are those that bind totumor-associated stroma. During tumor progression, the extracellularmatrix of the surrounding tissue is remodeled through two mainprocesses: the proteolytic degradation of extracellular matrixcomponents of normal tissue; and the de novo synthesis of extracellularmatrix components by tumor cells and stromal cells activated bytumor-induced cytokines. These two processes generate a “tumorextracellular matrix” or “tumor stroma”, which is permissive for tumorprogression and is qualitatively and quantitatively distinct from theextracellular matrices or stroma of normal tissues.

[0878] The “tumor stroma” thus has targetable components that are notpresent in formal tissues. Certain preferred tumor stromal targetingagents for use in the invention are those that bind to basement membranemarkers, type IV collagen, laminin, heparan sulfate, proteoglycan,fibronectins, activated platelets, LIBS, RIBS and tenascin. Thefollowing patents are specifically incorporated herein by reference forthe purposes of even further supplementing the present teachingsregarding the preparation and use of tumor stromal targeting agents:U.S. Pat. Nos. 5,877,289; 6,093,399; 6,004,555; and 6,036,955.

[0879] Components of tumor-associated stroma include structural andfunctional components of the stroma, extracellular matrix and connectivetissues. Tumor stroma targeting agents thus include those that bind tocomponents such as basement membrane markers, type IV collagens,laminin, fibrin, heparan sulfate, proteoglycans, glycoproteins, anionicpolysaccharides such as heparin and heparin-like compounds andfibronectins.

[0880] Exemplary useful antibodies are those that bind to tenascin, alarge molecular weight extracellular glycoprotein expressed in thestroma of various benign and malignant tumors. Anti-tenascin antibodiesmay thus be used as targeting agents (U.S. Pat. Nos. 6,093,399 and6,004,555, specifically incorporated herein by reference).

[0881] Further suitable targeting agents include antibodies and ligandsthat bind to a smooth muscle cell, a pericyte, a fibroblast, amacrophage, and an infiltrating lymphocyte or leucocyte. “Activatedplatelets” are further components of tumor stroma, as platelets bind tothe stroma when activated, and such platelets may thus be targeted bythe invention.

[0882] Further suitable stromal targeting agents, antibodies and antigenbinding regions thereof bind to “inducible” tumor stroma components,such as those inducible by cytokines, and especially those inducible bycoagulants, such as thrombin. A group of preferred anti-stromalantibodies are those that bind to RIBS, the receptor-induced bindingsite, on fibrinogen. “RIBS” is thus a targetable antigen, the expressionof which in stroma is dictated by activated platelets. Antibodies thatbind to LIBS, the ligand-induced binding site, on activated plateletsare also useful.

[0883] Particularly preferred targetable elements of tumor-associatedstroma are currently the tumor-associated fibronectin (FN) isoforms.Fibronectins are multifunctional, high molecular weight glycoproteinconstituents of both extracellular matrices and body fluids. They areinvolved in many different biological processes, such as theestablishment and maintenance of normal cell morphology, cell migration,haemostasis and thrombosis, wound healing and oncogenic transformation.

[0884] Fibronectin isoforms are ligands that bind to the integrin familyof receptors. “Tumor-associated fibronectin isoforms” may be consideredto be part of the tumor vasculature and/or the tumor stroma. Fibronectinisoforms have extensive structural heterogeneity, which is brought aboutat the transcriptional, post-transcriptional and post-translationallevels.

[0885] Structural diversity in fibronectins is first brought about byalternative splicing of three regions (ED-A, Ed-B and IIICS) of theprimary fibronectin transcript to generate at least 20 differentisoforms. As well as being regulated in a tissue- anddevelopmentally-specific manner, it is known that the splicing patternof fibronectin-pre-mRNA is deregulated in transformed cells and inmalignancies. In fact, the fibronectin isoforms containing the ED-A,ED-B and IIICS sequences are expressed to a greater extent intransformed and malignant tumor cells than in normal cells.

[0886] In particular, the fibronectin isoform containing the ED-Bsequence (B+ isoform), is highly expressed in foetal and tumor tissuesas well as during wound healing, but restricted in expression in normaladult tissues. B+ fibronectin molecules are undetectable in maturevessels, but upregulated in angiogenic blood vessels in normalsituations (e.g., development of the endometrium), pathologicalangiogenesis (e.g., in diabetic retinopathy) and in tumor development.The so-called B+ isoform of fibronectin (B-FN) is thus particularlysuitable for use with the present invention.

[0887] The ED-B sequence is a complete type III-homology repeat encodedby a single exon and comprising 91 amino acids. The presence ofB+isoform itself constitutes a tumor-induced neoantigen, but inaddition, ED-expression exposes a normally cryptic antigen within thetype III repeat 7 (preceding ED-B); since this epitope is not exposed infibronectin molecules lacking ED-B, it follows that ED-B expressioninduces the expression of neoantigens both directly and indirectly. Thiscryptic antigenic site forms the target of the monoclonal antibody, BC-1(European Collection of Animal Cell Cultures, Porton Down, Salisbury,UK, number 88042101). The BC1 antibody may be used as a vasculartargeting component of the present invention.

[0888] Improved antibodies with specificity for the ED-B isoform aredescribed in WO 97/45544, specifically incorporated herein by reference.Such antibodies have been obtained as single chain Fvs (scFvs) fromlibraries of human antibody variable regions displayed on the surface offilamentous bacteriophage (see also WO 92/01047, WO 92/20791, WO93/06213, WO 93/11236 and WO 93/19172).

[0889] Using an antibody phage library, specific scFvs can be isolatedboth by direct selection on recombinant fibronectin-fragments containingthe ED-B domain and on recombinant ED-B itself when these antigens arecoated onto a solid surface (“panning”). These same sources of antigenhave also been successfully used to produce “second generation” scFvswith improved properties relative to the parent clones in a process of“affinity maturation”. The isolated scFvs react strongly andspecifically with the B+ isoform of human fibronectin, preferablywithout prior treatment with N-glycanase.

[0890] The antibodies of WO 97/45544 are thus particularly contemplatedfor use herewith. In anti-tumor applications, these human antibodyantigen-binding domains are advantageous as they have less side-effectsupon human administration. The referenced antibodies bind the ED-Bdomain directly. Preferably, the antibodies bind both human fibronectinED-B and a non-human fibronectin ED-B, such as that of a mouse, allowingfor testing and analysis in animal models. The antibody fragments extendto single chain Fv (scFv), Fab, Fab′, F(ab′)₂, Fabc, Facb and diabodies.

[0891] Even further improved antibodies specific for the ED-domain offibronectin have been produced with sub-nanomolar dissociationconstants, as described in WO 99/58570, and are thus even more preferredfor use herewith. These targeting agents are exemplified by the L19antibody, described in WO 99/58570, specifically incorporated herein byreference for the purpose of teaching how to make and use this andrelated antibodies. These antibodies have specific affinity for acharacteristic epitope of the ED-B domain of fibronectin and haveimproved affinity to the ED-B epitope.

[0892] Such improved recombinant antibodies are available in scFvformat, from an antibody phage display library. In addition to H10 andL19, the latter of which has a dissociation constant for the ED-B domainof fibronectin in the sub-nanomolar concentration range, the techniquesof WO 99/58570, specifically incorporated herein by reference, may beused to prepare like antibodies. The isolation of human scFv antibodyfragments specific for the ED-B domain of fibronectin from antibodyphase-display libraries and the isolation of a human scFv antibodyfragment binding to the ED-B with sub-nanomolar affinity areparticularly described in Examples 1 and 2 of WO 99/58570.

[0893] Preferred antibodies thus include those with specific affinityfor a characteristic epitope of the ED-B domain of fibronectin, whereinthe antibody has improved affinity for the ED-B epitope, wherein theaffinity is in the subnanomolar range, and wherein the antibodyrecognizes ED-B(+) fibronectin. Other preferred formats are wherein theantibody is a scFv or recombinant antibody and wherein the affinity isimproved by introduction of a limited number of mutations in its CDRresidues. Exemplary residues to be mutated include 31-33, 50, 52 and 54of the VH domain and residues 32 and 50 of its VL domain. Suchantibodies are able to bind the ED-B domain of fibronectin with a Kd of27 to 54 pM; as exemplifed by the L19 antibody or functionallyequivalent variants form of L19.

[0894] Q5. Anti-Viral Conjugates

[0895] Under normal conditions, PE is not exposed at the cell surface.However, in various disease states, PE becomes exposed at the cellsurface of one or more cell types. For example, endothelial cells withintumor vasculature become PE-positive and can be targeted by PE-directedtherapeutics, as shown herein by the successful tumor treatment usingduramycin conjugated to HuIgG. PE also becomes exposed at the cellsurface of virally infected cells, which are thus additional targets fortherapeutic intervention using the PE-binding peptide derivatives of thepresent invention. Indeed, the present application shows that duramycinderivatives, as exemplified by those linked to biotin and HuIgG, areeffective anti-viral agents, both in vitro and in vivo.

[0896] Several anti-viral drugs, including AZT, acyclovir, gancyclovir,cidofovir (cytosine derivative) and new anti-viral drugs are limited bytoxicity/efficacy. Based on their observations regarding changes in PEduring viral infection, and further in light of the effectiveness of theoriginal PE-binding peptide derivatives, the present inventors haveaddressed problems in the anti-viral field by designing new anti-viraltherapeutics with reduced toxicity and increased efficacy. In the newanti-viral therapeutics of the invention, anti-viral drugs are linked toPE-binding peptides, which function to deliver the attached anti-viraldrugs to virally infected cells.

[0897] In addition, the inventors have the following observations inregard to the development of the PE-binding peptide, anti-viralderivatives of the present invention. Data are presented herein to showthat PE-binding peptide derivatives, e.g., duramycin-L-biotin, are takenup by macrophages in vivo, especially in the lung, even after systemicadministration. On infection, many viruses first pass through cells ofthe reticuloendothelial cell system (RES), and the macrophage is themain cell for viral uptake. Therefore, by linking anti-viral drugs toPE-binding peptides such as duramycin, the anti-viral effect of the drugis directed to the primary cell type (macrophage) responsible forclearing invading viruses.

[0898] As the PE-binding peptide derivatives localize to macrophages inthe lung after systemic administration will naturally be effective.Administration to the lung by more direct means, including via aerosol,is also envisioned. The present invention therefore solves importantdeficiencies in the viral treatment field by providing widely applicableand practical anti-viral remedies.

[0899] The new anti-viral therapeutics of the present invention thuscomprise a PE-binding peptide, such as duramycin, linked to ananti-viral drug, preferably using a biologically releasable orhydrolytically labile bond to link the two agents. Any of a range ofanti-viral agents, including any agent developed as an anti-viral in thefuture, may be linked to a PE-binding peptide to form an advantageousanti-viral therapeutic in accordance with this invention. In addition toso-called classic anti-viral agents, other DNA/RNA inhibitors may alsobe attached to a PE-binding peptide to form an anti-viral therapeutic.Exemplary anti-viral agents are listed in Table G, any one or more ofwhich may be attached to a PE-binding peptide to prepare an anti-viralconjugate of the invention, or can be used separately in the anti-viralcombination therapies of the invention. TABLE G Common Disease-CausingViruses and Anti-Viral Drugs Disease-Causing Viruses Drug CategoriesExemplary Anti-Viral Drugs Herpes virus Cidofovir, acyclovir,penciclovir (famciclovir), gancyclovir (ganciclovir), deoxyguanosine,foscarnet, idoxuridine, trifluorothymidine, vidarabine, sorivudineRetroviruses Nucleoside reverse Zidovudine, didanosine, transcriptase(RT) zalcitabine, lamivudine, inhibitors stavudine, abacavir,multinucleoside resistance A, multinucleoside resistance BNon-nucleoside RT Nevirapine, delavirdine, efavirenz, inhibitorsAdefovir Dipivoxil Protease Inhibitors Indinavir, ritonavir, saquinavir,nelfinavir, amprenavir Cell cycle phase specific Hydroxyurea (Hydrea ™,Bristol antineoplastic Myers-Squibb) Hepatitis B Deoxycytosineiphosphate, lamivudine triphosphate, emticitabine triphosphate, adefovirdiphosphate, penciclovir triphosphate, lobucavir triphosphate HepatitisC Interferon alpha, ribavirin Influenza A and B Amantadine, rimantadine,zanamivir, oseltamivir

[0900] Within the range of anti-viral agents and drugs, AZT andcidofovir are currently preferred for linking to a PE-binding peptide.Irrespective of the chosen anti-viral drug, the PE-binding peptide,anti-viral conjugate will bind to macrophages in the lungs, to virallyinfected cells and may also bind to virus particles. Depending on thelinker or conjugation technology used, the anti-viral drug may bereleased at the surface of the target cell and then be taken up into thecell. Preferably, the conjugate itself is taken up into the cell, suchas a macrophage or virally infected cell. Uptake can either occurnaturally or can be virus-mediated. Once inside the cell, as with anantibody conjugate, hydrolysis of the linker releases the activeanti-viral agent.

[0901] One example of a suitable linkage option for aduramycin-cidofovir anti-viral agent is set forth in FIG. 13R. In thisexample, the duramycin-cidofovir conjugate is designed to bind tomacrophages in the lungs and be taken into the cell. Hydrolysis of thelinker leads to decomposition of phosphoramidate and release of activecidofovir or a cell permeable derivative (R in FIG. 13R) that breaksdown to cidofovir.

[0902] Other linkages containing biologically labile bonds can be used,such as, e.g., disulfide, acid labile, enzymatically cleavable orhydrolysable. Accordingly, any biologically-releasable or selectivelyhydrolyzable bond described for use in linking antibodies to therapeuticagents can be used in connection with the PE-binding peptide, anti-viralderivatives of the present invention. The choice of linker is notlimited by the particular PE-binding peptide, such as duramycin, as thepeptide can be derivatized to introduce functional groups permitting theattachment of the selected anti-viral agent, as described above.

[0903] R. Anti-Viral Treatment Methods

[0904] The present invention further provides a range of antibodies,immunoconjugates and PE-binding peptide derivatives, optionallyconjugated to anti-viral agents, for use in treating viral infections.The treatment regimens, and particularly the doses, are generally asdescribed above for the cancer treatment aspects of the presentinvention, which adaptability is an advantage of the invention overall.Although an understanding of the particular mechanism(s) of action isnot necessary to practice the anti-viral treatment of the invention,certain of the reasons underlying the viral treatment, as supported bythe working examples herein, are as follows.

[0905] The most important mechanisms are believed to be connected withviral replication and activation of the host cell. During viralinfection, the virus activates the cell during its replication processinside the cell. This process of cell activation is necessary for viralreplication, as shown for herpes viruses, hepatitis C and HIV-1. Viralprogression activates gene expression, both viral and host. For example,the replication of Pichinde virus and Machupo virus is inhibited byactinomycin D late in the replication cycle, indicating that host cellgene transcription is needed for completion of viral replication.

[0906] The activation of the host cell by the virus causes the cell toexternalize anionic phospholipids and aminophospholipids, such PS andPE. In particular, the inventors reason that viral activation causesCa²⁺ fluxes into the cell, which activate scramblase, externalizinganionic phospholipids and aminophospholipids, particularly PS and PE.Antibodies, peptide derivatives and conjugates that bind anionicphospholipids and aminophospholipids, preferably PS and PE, then bindand interfere with the activation process, preventing the virus frombeing able to replicate properly.

[0907] The present examples show that the invention acts late in theprocess of viral infection, blocking viral maturation or egress. Theinventors' studies show that the inhibitory effect of the agents of theinvention is widely applicable, as it has been shown to operate onviruses that use different egression mechanisms. For example, thepresent examples demonstrate block of herpes virus (CMV), which escapesfrom Golgi-derived exocytotic vesicles, and block of arenavirus(Pichinde virus) and paramyxovirus (RSV), which bud out directly fromthe plasma membrane.

[0908] Virally infected cells externalize anionic phospholipids andaminophospholipids, particularly PS and PE, which are normallyintracellular, i.e., confined to the inner surface of plasma membrane.During escape of the virus, phospholipids redistribute at the site ofescape, accommodating membrane bending during viral budding orexocytosis from the plasma membrane, and anionic phospholipids andaminophospholipids are externalized during this process. The antibodies,peptide derivatives and conjugates of the invention can thus bind to theexternalized anionic phospholipids and aminophospholipids, particularlyPS and PE, and block the escape of the virus from the infected cell.Binding of the constructs of the invention to virally infected cells isalso shown in the present examples.

[0909] The antibodies, peptide derivatives and conjugates of theinvention may further bind to the externalized anionic phospholipids andaminophospholipids, particularly PS and PE, and interfere with one ormore signaling pathways necessary for viral gene expression and/orreplication.

[0910] Moreover, enveloped virions themselves likely have anionicphospholipids and aminophospholipids, such as PS and PE, on theirexternal surface. Since viruses lack a translocase to maintain orrestore phospholipid asymmetry, continued exposure of phospholipids suchas PS and PE is expected. The antibodies, peptide derivatives andconjugates of the invention may thus cause opsonization, complementbinding, phagocytosis by host cells such as macrophages and clearance offree virus particles.

[0911] In a further aspect of the invention, viruses likely need anionicphospholipids and aminophospholipids for infection and/or syncitiaformation. The antibodies, peptide derivatives and conjugates of theinvention may further block these aspects of the viral life cycle bybinding to anionic phospholipids and aminophospholipids.

[0912] According to the foregoing insights, and in light of the presentexamples, the spectrum of viral treatment for the present inventionextends to any virus, whether enveloped or not, DNA or RNA. As theanionic phospholipid- and aminophospholipid-binding antibodies, peptidederivatives and conjugates of the invention at least in part block viralreplication inside the cell, and/or prevent escape of virus from cells,the invention is not limited to the treatment of enveloped virusesalone, nor to any particular virus, which is an important advantage. Forexample, work published subsequent to the invention reports that annexinV and PS vesicles can inhibit HIV-1 infection of macrophages, but cannotinhibit HIV-1 infection of T cells or inhibit other viruses, such asvesicular stomatitis virus G and amphotropic murine leukemia virus(Callahan et al., 2003).

[0913] Naturally, the antibodies, peptide derivatives and conjugates ofthe invention do act on enveloped viruses, particularly those virusesthat have anionic phospholipids and aminophospholipids, of PS and PE, onthe outer surface of the envelope, wherein the antibodies, peptidederivatives and conjugates cause viral clearance and/or inhibiting viralentry of target cells.

[0914] An important aspect of the present invention is therefore that itis universally applicable, being suitable for the treatment ofrecombinant, engineered and synthetic viruses, e.g., created as part ofbio-terrorism. Indeed, the invention is not limited to the treatment ofanimals and humans. As the categories of hosts found in the virus taxainclude algae, archaea, bacteria, fungi, invertebrates, mycoplasma,plants, protozoa, spiroplasma and vertebrates, the invention can be usedto inhibit viral infection and replication in any such setting,including in viruses of agricultural importance. The treatment of viralinfection and associated diseases in vertebrates is currently preferred,and any one or more of the viruses in Table H, which infect vertebrateanimals, may be inhibited, and the resultant infection treated, usingthe present invention. TABLE H Viruses of Vertebrates Family Genus TypeSpecies Adenoviridae Mastadenovirus Human adenovirus 2 AviadenovirusFowl adenovirus 1 African Swine Fever-like Viruses African swine fevervirus Arenaviridae Arenavirus Lymphocytic choriomeningitis virusArterivirus Equine arteritis virus Astroviridae Astrovirus Humanastrovirus 1 Birnaviridae Aquabirnavirus Infectious pancreatic necrosisvirus Avibirnavirus Infectious bursal disease virus BunyaviridaeBunyavirus Bunyamwera virus Hantavirus Hantaan virus Nairovirus Nabrobisheep disease virus Phlebovirus Sandfly fever Sicilian virusCaliciviridae Calicivirus Vesicular exanthema of swine virusCircoviridae Circovirus Chicken anemia virus Coronaviridae CoronavirusAvian infectious bronchitis virus Torovirus Berne virus DeltavirusHepatitis delta virus Filoviridae Filovirus Marburg virus FlaviviridaeFlavivirus Yellow fever virus Pestivirus Bovine diarrhea virus HepatitisC —like viruses Hepatitis C virus Hepadnaviridae OrthophepadnavirusHepatitis B virus Avihepadnavirus Duck hepatitis B virus HerpesviridaeSubfamily Alphaherpesvirinae Simplexvirus Human herpesvirus 1Varicellovirus Human herpesvirus 3 Subfamily: BetaherpesvirinaeCytomegalovirus Human herpesvirus 5 Muromegalovirus Mousecytomegalovirus 1 Subfamily Gammaherpesvirinae Roseolovirus Humanherpesvirus 6 Lymphocryptovirus Human herpesvirus 4 Rhadinovirus Atelineherpesvirus 2 Iridoviridae Ranavirus Frog virus 3 LymphocystivirusFlounder virus Goldfish virus —like viruses Goldfish virus 1Orthomyxoviridae Influenzavirus A, B Influenza A virus Influenzavirus CInfluenza C virus Thogoto-Like viruses Thogoto virus PapovaviridaePolyomavirus Murine polyomavirus Papillomavirus Cottontail rabbitpapillomavirus (Shope) Paramyxoviridae Subfamily ParamyxovirinaeParayxovirus Human parainfluenza virus 1 Morbillivirus Measles virusRubulavirus Mumps virus Subfamily Pneumovirinae Pneumovirus Humanrespiratory syncytial virus Parvoviridae Subfamily ParovirinaeParvovirus Mice minute virus Erythovirus B19 virus DependovirusAdeno-associated virus 2 Picornaviridae Enterovirus Poliovirus 1Rhinovirus Human rhinovirus 1A Hepatovirus Hepatitis A virus CardiovirusEncephalomyocarditis virus Aphthovirus Foot-and-mouth disease virus OPoxviridae Subfamily Chordopoxvirinae Orthopoxvirus Vaccinia virusParapoxyvirus Orf virus Avipoxvirus Fowlpox virus Capripoxvirus Sheeppoxvirus Leporipoxvirus Myxoma virus Suipoxvirus Swinepox virusMolluscipoxvirus Molluscum contagiosum virus Yatapoxvirus Yaba monkeytumor virus Reoviridae Orthoreovirus Reovirus 3 Orbivirus Bluetonguevirus 1 Rotavirus Simian rotavirus SA11 Coltivirus Colorado tick fevervirus Aquareovirus Golden shiner virus Retroviridae Mammalian type Bretroviruses Mouse mammary tumor virus Mammalian type C retrovirusesMurine leukemia virus Avian type C retroviruses Avian leukosis virusType D retroviruses Mason-Pfizer monkey virus Blv-htlv retrovirusesBovine leukemia virus Lentivirus Human immunodeficiency virus 1Spumavirus Human spumavirus Rhabdoviridae Vesiculovirus Vesicularstomatitis Indiana virus Lyssavirus Rabies virus Ephemerovirus Bovineephemeral fever Togaviridae Alphavirus Sindbis virus Rubivirus Rubellavirus

[0915] The use of the invention in treating viral infections andassociated diseases in mammals is preferred, particularly in terms ofvaluable or valued animals, such as racehorses and domestic pets, andanimals and birds used to directly produce (e.g., meat) or indirectlyproduce (e.g., milk and eggs) food for human consumption. In addition tohuman treatment. exemplary embodiments of the invention include thetreatment of horses, dogs, cats and the like; the treatment of cows,pigs, boar, sheep, goat, buffalo, bison, llama, deer, elk, and otherlarge animals, as well as their young, including calves and lambs.

[0916] The treatment of humans is particularly preferred, whether fornaturally occurring viruses or for those created by bioterrorism. Interms of naturally occurring viruses and the resultant diseases, theinvention is again unlimited in its applications. Accordingly, any oneor more of the viruses in Table J may be inhibited using the presentinvention, and the resultant infections and diseases thus treated. TABLEJ Viral Diseases in Humans Disease Virus Type of Virus AIDS HumanImmunodeficiency Retrovirus Virus (HIV) Bronchiolitis and viralpneumonia Respiratory syncytial virus Paramyxovirus BronchiolitisParainfluenza virus Paramyxovirus Cervical cancer Human papilloma virusPapovavirus Chicken pox Varicella Zoster virus Herpesvirus Dengue Denguevirus Flavivirus Ebola hemorrhagic fever Ebola virus Filovirus GenitalHerpes Herpes Simplex virus-2 Herpesvirus Hantavirus hemorrhagic feverHantavirus Bunyavirus Hepatitis Hepatitis A Picornavirus Hepatitis BHepadavirus Hepatitis C Flavivirus Hepatitis D Deltavirus Hepatitis ECalcivirus Influenza Influenza viruses A, B and C Orthomyxovirus JuninArgentinian Hemorrhagic Fever Junin virus Arenavirus Lassa hemorrhagicfever Lassa virus Arenavirus Machupo hemorrhagic fever Machupo virusArenavirus Measles Rubeola virus Paramyxovirus Mononucleosis EpsteinBarr virus Herpesvirus CMV disease (viral pneumonia. CytomegalovirusHerpesvirus mononucleosis like syndrome) Severe Acute RespiratorySyndrome Human coronavirus Coronavirus (SARS) Shingles Varicella zostervirus Herpesvirus Smallpox Variola virus Poxvirus Yellow fever Yellowfever virus Flavivirus West Nile Disease West Nile virus Western equineencephalitis Western EE virus Togavirus Pneumonia, Hepatitis, acuteAdenovirus Adenovirus respiratory disease Gastroenteritis RotavirusRotavirus Encephalitis Semliki Forest virus Alphavirus Cowpox Vacciniavirus Poxvirus Encephalitis Venezuelan EE Alphavirus Meningitis,encephalitis, Lymphocytic choriomeningitis Arenavirusmeningoencephalitis Venezuelan hemorrhagic fever Guanarito virusArenavirus Rift valley fever (hemorrhagic fever, Rift valley fever virusBunyavirus encephalitis) Marburg Hemorrhagic fever Marburg virusFilovirus Tick borne encephalitis Tick borne encephalitis virusFlavivirus (TBEV) Encephalitis Hendra virus Paramyxovirus EncephalitisNipah virus Paramyxovirus Crimean-Congo hemorrhagic fever Crimean-Congohemorrhagic Bunyavirus fever virus Brazilian hemorrhagic fever Sabiavirus Arenavirus

[0917] The invention is particularly contemplated for use in thetreatment of CMV related diseases such as viral pneumonia, mononucleosislike syndrome, and associated congenital malformations (deafness andmental retardation); respiratory diseases, such as those caused by RSV,including bronchiolitis and viral pneumonia, influenza, the common coldand SARS: AIDS; hepatitis; cancers associated with viral infections;mononucleosis; and smallpox.

[0918] In other embodiments, the inventors particularly contemplate theinhibition of arenaviruses, which are pathogenic in man. Thearenaviruses include the Old World viruses responsible for Lassa fever(Lassa virus) and lymphocytic choriomeningitis (LCMV). Lassa fever isendemic in West Africa, affecting up to 300,000 people annually andcausing up to 3000 deaths. Infection with Lassa fever leads to fever andmalaise within about 10 days. Abdominal pain, nausea, vomiting anddiarrhea are common. Pharyngitis and cough may develop. Neurologicalsymptoms are usually mild. Vascular leak syndromes, such as edema andpleural effusions, are present in more severe cases. Bleeding is seenabout one quarter of patients. The disease can cause changes in thecardiovascular system that culminate in shock and death.

[0919] Arenaviruses also include and the antigenically-distinct NewWorld viruses responsible for Argentine hemorrhagic fever (Junin virus),Bolivian hemorrhagic fever (Machupo virus) and Venezuelan hemorrhagicfever (Guanarito virus). All of these viruses are on the CDC Category Alist of potential bioterrorism weapons.

[0920] Although not connected with aminophospholipids or anionicphospholipids, other antibodies that bind to viruses directly have beendeveloped into approved drugs. This is true of CytoGam, which is usedfor suppressing CMV infections in immunosuppressed patients, andSynagis, which is used to protect newborn infants from respiratorysyncitial virus. Thus, there are no problems in the use of monoclonalantibodies to access and neutralize viruses in tissues.

[0921] The doses that are suitable for the anti-tumor embodiments arealso suitable for the anti-viral treatments. Similarly, multipleadministration may be used for chronic infections, and high doses may beused for acute infections. Any suitable route of administration may beemployed, again as disclosed for the cancer treatment aspects, includingIV, IM, SC, as an aerosol to lungs or airways and such like.

[0922] The therapeutics provided by the invention are valuable agentshaving broad-spectrum anti-viral activity. In addition to beingeffective against a large number of potentially lethal viruses, theagents an also be administered after exposure to the virus, even insettings where the exact nature of the virus is not known. Thus, theanti-viral therapeutics of the present invention do not require aprolonged period of time between identification of the pathogen anddelivery of the therapy, in marked contrast with the time and expenseentailed by the development, production or delivery of specificvaccines.

[0923] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE I Tumor Treatment with Anti-VCAM-1-tTF Coaguligand

[0924] The present example shows the specific coagulation of tumorvasculature in vivo that results following the administration of a tumorvasculature-targeted coagulant (“coaguligand”) to tumor-bearing animalsand the resultant anti-tumor effects. In this coaguligand, an antibodydirected to VCAM-1 (vascular endothelial adhesion molecule-1, VCAM-1) isused as a targeting agent to deliver truncated Tissue Factor (tTF), amodified form of a human coagulant, to tumor vasculature.

[0925] The MK2.7 hybridoma, secreting a rat IgG₁ antibody against murineVCAM-1, was obtained from the American Type Culture Collection (ATCC,Rockville, Md.; ATCC CRL 1909). The R187 hybridoma, secreting a rat IgG₁antibody against murine viral protein p30 gag, was also obtained fromthe ATCC, and was used as an isotype matched control for the anti-VCAM-1antibody.

[0926] The blood vessels of the major organs and a tumor from micebearing subcutaneous L540 human Hodgkin's tumors were examinedimmunohistochemically for VCAM-1 expression using an anti-VCAM-1antibody. Overall, VCAM-1 expression was observed on 20-30% of totaltumor blood vessels stained by the anti-endoglin antibody, MJ 7/18, usedas a positive control. Constitutive vascular expression of VCAM-1 wasfound in heart and lungs in both tumor-bearing and normal animals.Strong stromal staining was observed in testis where VCAM-1 expressionwas strictly extravascular.

[0927] Mice bearing subcutaneous L540 tumors were injected intravenouslywith anti-VCAM-1 antibody and, two hours later, the mice wereexsanguinated. The tumor and normal organs were removed and frozensections were prepared and examined immunohistochemically to determinethe location of the antibody. Anti-VCAM-1 antibody was detected onendothelium of tumor, heart and lung. Staining was specific as nostaining of endothelium was observed in the tumor and organs of miceinjected with a species isotype matched antibody of irrelevantspecificity, R187. No localization of anti-VCAM-1 antibody was found intestis or any normal organ except heart and lung.

[0928] An anti-VCAM-1•tTF conjugate or “coaguligand” was prepared usingtruncated tissue factor (tTF). Intravenous administration of theanti-VCAM-1•tTF coaguligand induces selective thrombosis of tumor bloodvessels, as opposed to vessels in normal tissues, in tumor-bearing mice.

[0929] The anti-VCAM-1•tTF coaguligand was administered to mice bearingsubcutaneous L540 tumors of 0.4 to 0.6 cm in diameter. Beforecoaguligand injection, tumors were healthy, having a uniform morphologylacking regions of necrosis. The tumors were well vascularized and had acomplete absence of spontaneously thrombosed vessels or hemorrhages.Within four hours of coaguligand injection, 40-70% of blood vessels werethrombosed, despite the initial staining of only 20-30% of tumor bloodvessels. The thrombosed vessels contained occlusive platelet aggregates,packed erythrocytes and fibrin. In several regions, the blood vesselshad ruptured, spilling erythrocytes into the tumor interstitium.

[0930] By 24 h after coaguligand injection, the blood vessels were stilloccluded and extensive hemorrhage had spread throughout the tumor. Tumorcells had separated from one another, had pyknotic nuclei and wereundergoing cytolysis. By 72 h. advanced necrosis was evident throughoutthe tumor. It is likely that the initial coaguligand-induced thrombindeposition results in increased induction of the VCAM-1 target antigenon central vessels, thus amplifying targeting and tumor destruction.

[0931] The thrombotic action of anti-VCAM-1•tTF on tumor vessels wasantigen specific. None of the control reagents administered atequivalent quantities (tTF alone, anti-VCAM-1 antibody alone. tTF plusanti-VCAM-1 antibody or the control coaguligand of irrelevantspecificity) caused thrombosis.

[0932] In addition to the thrombosis of tumor blood vessels, this studyalso shows that intravenous administration of the anti-VCAM-1•tTFcoaguligand does not induce thrombosis of blood vessels in normalorgans. Despite expression of VCAM-1 on vessels in the heart and lung ofnormal or L540 tumor-bearing mice, thrombosis did not occur afteranti-VCAM-1•tTF coaguligand administration. No signs of thrombosis,tissue damage or altered morphology were seen in 25 mice injected with 5to 45 μg of coaguligand 4 or 24 h earlier. There was a normalhistological appearance of the heart and lung from the same mouse thathad major tumor thrombosis. All other major organs (brain, liver,kidney, spleen, pancreas, intestine, testis) also had unalteredmorphology.

[0933] Frozen sections of organs and tumors from coaguligand-treatedmice gave coincident staining patterns when developed with either theanti-TF antibody, 10H10, or an anti-rat IgG antibody and confirmed thatthe coaguligand had localized to vessels in the heart, lung and tumor.The intensity of staining was equal to that seen when coaguligand wasapplied directly to the sections at high concentrations followed bydevelopment with anti-TF or anti-rat IgG, indicating that saturation ofbinding had been attained in vivo.

[0934] These studies show that binding of coaguligand to VCAM-1 onnormal vasculature in heart and lung is not sufficient to inducethrombosis, and that tumor vasculature provides additional factors tosupport coagulation.

[0935] The anti-tumor activity of anti-VCAM-1•tTF coaguligand wasdetermined in SCID mice bearing 0.3-0.4 cm³ L540 tumors. The drug wasadministered i.v. 3 times at intervals of 4 days. Mean tumor volume ofanti-VCAM-1•tTF treated mice was significantly reduced at 21 days oftreatment (P<0.001) in comparison to all other groups. Nine of a totalof 15 mice treated with the specific coaguligand showed more than 50%reduction in tumor volume. This effect was specific since unconjugatedtTF, control IgG coaguligand and mixture of free anti-VCAM-1 antibodyand tTF did not affect tumor growth.

EXAMPLE II Phosphatidylserine Expression on Tumor Blood Vessels

[0936] To explain the lack of thrombotic effect of anti-VCAM-1•tTF onVCAM-1 positive vasculature in heart and lungs, certain of the inventorsdeveloped a concept of differential aminophospholipid and anionicphospholipid, e.g. PS and PE, localization between normal and tumorblood vessels. Specifically, they hypothesized that endothelial cells innormal tissues segregate aminophospholipids and anionic phospholipids,e.g. PS and PE, to the inner surface of the plasma membrane phospholipidbilayer, where PS is unable to participate in thrombotic reactions:whereas endothelial cells in tumors translocate aminophospholipids andanionic phospholipids to the external surface of the plasma membrane,where PS can support the coagulation action of the coaguligand. PSexpression on the cell surface allows coagulation because it enables theattachment of coagulation factors to the membrane and coordinates theassembly of coagulation initiation complexes.

[0937] The inventors' model of aminophospholipid and anionicphospholipid translocation to the surface of tumor blood vesselendothelial cells, as developed herein, is surprising in that PSexpression does not occur after, and does not inevitably trigger, celldeath. Aminophospholipid and anionic phospholipid expression at thetumor endothelial cell surface is thus sufficiently stable to allowaminophospholipids and anionic phospholipids. e.g. PS and PE, to serveas targetable entities for therapeutic intervention.

[0938] To confirm the hypothesis that tumor blood vessel endotheliumexpresses PS on the luminal surface of the plasma membrane, theinventors used the following immunohistochemical study to determine thedistribution of anti-PS antibody after intravenous injection into L540tumor bearing mice.

[0939] A. Methods

[0940] Anti-PS and anti-cardiolipin antibodies, both mouse monoclonalIgM antibodies, were produced and characterized by Rote et al. (1993,incorporated herein by reference) as described in Example IV. The majorreactivity of 3SB is with PS, but it also has reactivity with theanionic phospholipid, phosphatidic acid, a relatively minor component ofthe plasma membrane also tightly segregated to the internal leaflet innormal cells.

[0941] L540 tumor-bearing mice were injected i.v. with 20 μg of eitheranti-PS or anti-cardiolipin mouse IgM antibodies. After 10 min., micewere anesthetized and their blood circulations were perfused withheparinized saline. Tumors and normal tissues were removed andsnap-frozen. Serial sections of organs and tumors were stained witheither HRP-labeled anti-mouse IgM for detection of anti-PS antibody orwith anti-VCAM-1 antibody followed by HRP-labeled anti-rat Ig.

[0942] To preserve membrane phospholipids on frozen sections, thefollowing protocol was developed. Animals were perfused with DPBScontaining 2.5 mM Ca²⁺. Tissues were mounted on3-aminopropyltriethoxysilane-coated slides and were stained within 24 h.No organic solvents, formaldehyde or detergents were used for fixationor washing of the slides. Slides were re-hydrated by DPBS containing 2.5mM Ca²⁺ and 0.2% gelatin. The same solution was also used to washsections to remove the excess of reagents. Sections were incubated withHRP-labeled anti-mouse IgM for 3.5 h at room temperature to detectanti-PS IgM.

[0943] B. Results

[0944] This immunohistochemical study showed that anti-PS antibodylocalized within 10 min. to the majority of tumor blood vessels,including vessels in the central region of the tumor that can lackVCAM-1. Vessels that were positive for VCAM-1 were also positive for PS.Thus, there is coincident expression of PS on VCAM-1-expressing vesselsin tumors.

[0945] In the in vivo localization studies, none of the vessels innormal organs, including VCAM-1-positive vasculature of heart and lung,were stained, indicating that PS is absent from the external surface ofthe endothelial cells. In contrast, when sections of normal tissues andtumors were directly stained with anti-PS antibody in vitro, nodifferences were visible between normal and tumor, endothelial or othercell types, showing that PS is present within these cells but onlybecomes expressed on the surface of endothelial cells in tumors.

[0946] The specificity of PS detection was confirmed by two independentstudies. First, a mouse IgM monoclonal antibody directed against adifferent negatively charged lipid, cardiolipin, did not home to tumoror any organs in vivo. Second, pretreatment of frozen sections withacetone abolished staining with anti-PS antibody, presumably because itextracted the lipids together with the bound anti-PS antibody.

EXAMPLE III Annexin V Blocks Coaguligand Activity

[0947] The present example provides further evidence of the role ofsurface PS expression in coaguligand activity from studies using thehigh affinity PS binding ligand, annexin V, to block PS function invitro and in vivo.

[0948] A. Annexin V Blocks Coaguligand Activation of Factor X In Vitro

[0949] The ability of Annexin V to affect Factor Xa formation induced bycoaguligand was determined by a chromogenic assay. IL-1α-stimulatedbEnd.3 cells were incubated with anti-VCAM-•tTF and permeabilized bysaponin. Annexin V was added at concentrations ranging from 0.1 to 10μg/ml and cells were incubated for 30 min. before addition of dilutedProplex T. The amount of Factor Xa generated in the presence or absenceof Annexin V was determined. Each treatment was performed in duplicateand repeated at least twice.

[0950] The need for surface PS expression in coaguligand action isfurther indicated by the inventors' finding that annexin V, which bindsto PS with high affinity, blocks the ability of anti-VCAM-1•tTF bound tobEnd.3 cells to generate factor Xa in vitro.

[0951] Annexin V added to permeabilized cells preincubated withanti-VCAM-1•tTF inhibited the formation of factor Xa in a dose-dependentmanner. In the absence of Annexin V, cell-bound coaguligand produced 95ng of factor Xa per 10,000 cells per 60 min. The addition of increasingamounts of Annexin V (in the μg per ml range) inhibited factor Xaproduction. At 10 μg per ml, Annexin V inhibited factor Xa production by58%. No further inhibition was observed by increasing the concentrationof Annexin V during the assay, indicating that annexin V saturated allavailable binding sites at 10 μg per ml.

[0952] B. Annexin V Blocks Coaguligand Activity In Vivo

[0953] The ability of Annexin V to inhibit coaguligand-inducedthrombosis in vivo was examined in L540 Hodgkin-bearing SCID mice.Tumors were grown in mice and two mice per group (tumor size 0.5 cm indiameter) were injected intravenously via the tail vein with one of thefollowing reagents: a) saline; b) 100 μg of Annexin V; c) 40 μg ofanti-VCAM-1•tTF; d) 100 μg of Annexin V followed 2 hours later by 40 μgof anti-VCAM-1•tTF.

[0954] Four hours after the last injection mice were anesthetized andperfused with heparinized saline. Tumors were removed, fixed with 4%formalin, paraffin-embedded and stained with hematoxylene-eosin. Thenumber of thrombosed and non-thrombosed blood vessels was counted andthe percentage of thrombosis was calculated.

[0955] Annexin V also blocks the activity of the anti-VCAM-1•tTFcoaguligand in vivo. Groups of tumor-bearing mice were treated with oneof the control or test reagents. The mice were given (a) saline; (b) 100μg of Annexin V; (c) 40 μg of anti-VCAM-1•tTF coaguligand; or (d) 100 μgof Annexin V followed 2 hours later by 40 μg of anti-VCAM-1•tTFcoaguligand. Identical results were obtained in both mice per group.

[0956] No spontaneous thrombosis, hemorrhages or necrosis were observedin tumors derived from saline-injected mice. Treatment with Annexin Valone did not alter tumor morphology.

[0957] In accordance with other data presented herein, 40 μg ofanti-VCAM-1•tTF coaguligand caused thrombosis in 70% of total tumorblood vessels. The majority of blood vessels were occluded with packederythrocytes and clots, and tumor cells were separated from one another.Both coaguligand-induced anti-tumor effects, i.e., intravascularthrombosis and changes in tumor cell morphology, were completelyabolished by pre-treating the mice with Annexin V.

[0958] These findings confirm that the anti-tumor effects ofcoaguligands are mediated through the blockage of tumor vasculature.These data also demonstrate that PS is essential for coaguligand-inducedthrombosis in vivo.

EXAMPLE IV Generation of Antibodies to Aminophospholipids and AnionicPhospholipids

[0959] This example describes an immunization protocol designed by theinventors in light of their observations on aminophospholipid andanionic phospholipid translocation in tumor vascular endothelial cells,and discovered to function well in the generation of antibodies againstaminophospholipids and anionic phospholipids. A number of antibodiesreactive with aminophospholipids and anionic phospholipids, such as PSand PE, were obtained. In the present and following examples, forsimplicity, antibodies reactive with PS can be termed “anti-PSantibodies”, although the binding of certain of these antibodies is notrestricted to PS but extends to certain other aminophospholipids andanionic phospholipids as shown herein.

[0960] A. Immunization Protocol

[0961] To present aminophospholipids and anionic phospholipids to theimmune system as stronger immunogens, the aminophospholipids and anionicphospholipids were formulated as aminophospholipid-positive and anionicphospholipid-positive cells. The membrane-inserted aminophospholipidsand anionic phospholipids, surrounded by other membrane components, havea better conformation and clearance rate for raising antibodies.

[0962] The intent is to immunize immunocompetent animals with autologouscells expressing aminophospholipids and anionic phospholipids, asexemplified in this instance by PS, wherein the animals would notproduce antibodies against all self surface antigens, but wouldrecognize membrane-exposed phospholipids, e.g. PS, as a foreign element.The procedure is applicable to the use of any standard laboratoryanimals, such as immunocompetent BALB/c mice and Lewis rats, with anyaminophospholipid-positive or anionic phospholipid-positive cells.

[0963] BALB/c mice and mouse endothelioma cells, bEnd.3 (immortalizedmouse (BALB/c strain) endothelial cells), were first chosen. bEnd.3 werecultured in 10% DMEM with 9 ml/500 ml HEPES Buffer, in 10% CO₂incubator. The bEnd.3 cells were expanded in T175 TC flasks until thedesired number of cells were obtained. Typically, each flask at ˜70-80%confluency has about 3×10⁶ cells, and each mouse should receive from1×10⁶ to 20×10⁶ cells, up to 1×10⁷ cells.

[0964] bEnd.3 cells are treated with 50 μM to 200 μM of hydrogenperoxide for 1 or 2 hours at 37° C. to expose anionic phospholipids,such as PS, before immunization. The stock of H₂O₂ is [9.8M]; 30% (v/v).This is diluted 1:1000, then 0.4 ml is add into the T175 TC flask with40 ml media to a final concentration of 100 μM H₂O₂. The cells weremaintained for 1 hour at 37° C. To harvest, the cells were washed 3×with warm PBS, +10 mM EDTA, to remove all BSA or serum protein in themedium. The cells were remove with gentle trypsin treatment, washed andcentrifuged for 5 minutes at 1000 rpm. The supernatant was aspirated andthe cells resuspended in DMEM without additives to the appropriatevolume (each mouse receives about 1×10⁷ cells in 200 μl) and kept onice.

[0965] Cells treated in this manner were injected (200 μl of cellsuspension) into each mouse IP using 1 ml syringe and 23 gauge needle.Mice were immunized from three to seven times at intervals of 3 to 4weeks. Immune sera were collected by bleeding the mice ten days aftereach boost, starting from the second boost. The serum titer for anti-PSwas tested by ELISA.

[0966] These immunizations with autologous PS-positive cells did notresult in unrestricted production of autoantibodies, but were limited tothe production of antibodies reactive with PS or reactive with PS incombination with other aminophospholipids and anionic phospholipids.

[0967] In another study, female Lewis rats were immunized with bEnd.3endothelial cells that had been treated with 200 μM of hydrogen peroxidefor 2 h. The treatment caused translocation of anionic phospholipids tothe external surface in 70-90% of cells as detected by ¹²⁵I-labeledannexin V. Treated cells were washed, detached and counted. Two millioncells were suspended in sterile PBS and injected 5 times i.p. with theinterval of 3 wk between injections. The titer of polyclonal antibodiesto anionic phospholipids was determined 2 days after each immunization.

[0968] B. High Titer Antisera

[0969] Mice with extremely high titers of antibodies reactive withanionic phospholipids such as PS were obtained (Table 1). The mice didnot show any signs of toxicity. Although this immunization protocol wasmore effective in mice than rats overall, immunization of rats waseffective and produced the 9D2 antibody (see below). TABLE 1 Anti-PS IgGAntibody Generation Number of Mice per Group Titer Range (% of total)   1:100-1:1,000  2/30 (6.66%)   1:1000-1:10,000  5/30 (16.6%) 1:10,000-1:100,000 18/30 (60%) 1:100,000-1,000,000  5/30 (16.6%)

[0970] In further immunizations, various mice were immunized three timeswith hydrogen peroxide-treated bEnd.3 cells and the serum was tested 54days after the first immunization. IgG antibodies reactive with PSwithin serum were detected with an anti-mouse IgG. Fc specific secondaryantibody, and IgM antibodies within serum were detected with ananti-mouse IgG mu specific secondary antibody. A number of effectiveantisera with IgG and IgM antibodies reactive with PS were obtainedusing this immunization protocol, of which the antisera with IgGantibodies were generally more effective.

[0971] These methods can now be used to generate further particularanti-PS antibodies. e.g., including those screened for effectivelycompetition with the 3G4 antibody described below. Typically, when theIgG titer of the desired antisera for PS reaches >200,000, but PC titeris <50,000, fusion can be performed to generate the monoclonal antibody.

[0972] Also, these methods are not limited to initial cell treatmentwith H₂O₂, as other methods to induce expression of aminophospholipidsand anionic phospholipids can be used. For example, treatment with TNFand actinomycin D is another useful method. In one case, subconfluent(˜85% confluence) bEnd. 3 cells were treated with 10 ng/ml mouse TNF and1 μg/ml actinomycin D for 16 hrs at 37° C. in the incubator. The cellswere then taken through the immunization procedure as outlined above.

[0973] C. IgG and IgM Monoclonal Antibodies

[0974] Hybridomas were obtained by fusing splenocytes from immunizedanimals with myeloma partner P3X63AG8.653 cells (ATCC, Rockville, Md.).

[0975] An important aspect of the inventors' technique to preparemonoclonal antibodies useful in tumor treatment is the selectionstrategy, which involves screening to select antibodies that bind toaminophospholipids or anionic phospholipids, but not to neutralphospholipids. Another important aspect is to select antibodies thatbind to PS-coated plates as strongly in the presence of serum as in theabsence of serum. This is carried out to exclude antibodies thatrecognize complexes of PS and serum proteins, which are believed tocause or contribute to anti-phospholipid syndrome.

[0976] The strategy to isolate monoclonal antibodies reactive with PS,for example, involved screening hybridoma supernatants on PS-coatedplates using an anti-mouse IgG, Fc gamma specific secondary antibody.Screening was first conducted against four phospholipids (PS,sphatidylserine; PE, phosphatidylethanolamine; CL, cardiolipin; and PC,phosphatidylcholine), as well as bEnd3 cells. Clones reactive with theneutral phospholipid, PC were discarded, as were clones non-reactivewith bEnd3 cells. High binding anti-PS clones were selected. The wellsthat had PS only reactivity, or strong preference for PS were sub-clonedfirst, and wells that exhibited PS reactivity in combination withbinding to other anionic phospholipids were sub-cloned second.

[0977] In certain in the following studies, mouse monoclonal IgMantibodies termed 3SB, D11 and BA3, produced as described by Rote et al.(1993), were also included. The 3SB antibody is described in theliterature as an anti-PS antibody and the D11 antibody is described inthe literature as an anti-cardiolipin (anti-CL) antibody. Details of thegeneration and characterization of these antibodies were reported byRote et al. (1993, incorporated herein by reference).

[0978] The isotype of each selected hybridoma generated by the inventorswas determined. As antibodies of IgG class have numerous advantages overIgM, including typically higher affinity, lower clearance rate in vivoand simplicity of purification, modification and handling, theirgeneration was particularly desired. To focus on wells with homogeneousIgG isotype, wells containing IgM or a mixture of different Igs werediscarded or re-cloned. Sub-cloning of highly positive clones wasrepeated three to four times.

[0979] The isotype of representative IgG and IgM antibodies, asdetermined by ELISA, is shown in Table 2. The inventors initially termedthe 3G4 antibody “F3-G4”, before changing the designation to 3G4. Thisdoes not reflect any change in biological material. The serum dependenceor independence of the antibodies is also set forth in Table 2. TABLE 2Isotype and Serum-Dependence of Anti-PS Antibodies Name OriginSpecies/Isotype Serum-dependence 3SB Rote et al., 1993 Mouse IgM kappaNone D11 N. Rote Mouse IgM kappa BA3 Rote et al., 1993 Mouse IgM kappa9D2 This study Rat IgM kappa None 1B12 This study Mouse IgG₁ kappa 3G4This study Mouse IgG₃ kappa None 1B9 This study Mouse IgG₁ kappaAbsolute 3B10 This study Mouse IgG₃ kappa None 2G7 This study Mouse IgG₁kappa Absolute 7C5 This study Mouse IgG₁ kappa Absolute

[0980] D. ELISA Protocol and Monoclonal Antibody Characterization

[0981] The antibodies were studied further by ELISA and compared to 3SBand D11. The anti-PS ELISA used in the present studies is conducted asfollows. Unless particular differences are specified, this is the formatof the ELISA used throughout the studies of the present application.

[0982] The ELISA is exemplified using the antigen PS (P-6641 25 mg 10mg/ml (solvent is Chloroform:MeOH 95:5) in 2.5 ml bottle). Otherphospholipids can be used using the same protocol. The PS (or otherphospholipids) stock solution should be aliquoted and stored in anairtight container at −30° C. The preferred 96 well plates are DynatechU bottom Immulon 1 (from Dynatech Labs. Cat#011-010-3550).

[0983] The standard blocking buffer used herein is 10% bovine serumdissolved in PBS. Other blocking solutions are suitable, but anydetergents should be excluded from block and wash solutions. The primaryantibody is the test sample or admixture. The preferred secondaryantibody is goat, anti-mouse IgG-HRP. The developing solutions are: 10ml of 0.2M Na₂PO₄, 10 ml of 0.1M citric acid, one 10 mg tablet of OPD,and 10 μl of hydrogen peroxide. The stop solution is 0.18 M H₂SO₄.

[0984] The protocol entails coating 96-well plate with PS as follows:dilute the PS stock solution in n-hexane to 10 μg/ml and mix well. Add50 μl to each well and allow this to evaporate for one hour. Add 200 μlof 10% serum (or other blocking buffer) to each well, cover and maintainat room temperature for 2 hours or overnight at 4° C. Wash the platethree times with PBS. Add the primary antibody (dilute in blockingbuffer) and incubate for 2 hours at 37° C. Wash three times with PBS.Add 100 μl/well of secondary antibody (typically goat, anti-mouseIgG-HRP or other appropriate secondary antibody) and incubate for 1 hourat 37° C. Wash the plate three times with PBS. Develop the ELISA byadding 100 μl of developing solution to each of the wells, develop for10 minutes, then add 100 μl of stop solution to each plate and read theO.D. at 490 nm.

[0985] The following results are presented for 9D2, 1B12, 3G4 and 1B9.The affinity of these antibodies for PS was determined and compared to3SB. Certain of the relative affinities of the new antibodies are muchimproved compared to 3SB (Table 3). TABLE 3 Relative Affinity of Anti-PSAntibodies EC₅₀ Binding vs. 3SB EC₅₀ Affinity vs. 3SB Name (μg/ml)¹(-fold increased) (nM)² (-fold increased) 3SB 0.468 1 0.518 1 D11 >40.00.011 >44.4 0.011 9D2 0.104 4.50 0.115 4.50 1B12 0.312 1.50 2.07 0.253G4 0.040 11.7 0.266 1.94 1B9 0.019 24.6 0.126 4.11 Annexin V³ 0.1004.68 2.77 0.18

[0986] The specificity of the antibodies was determined by ELISA usingplates coated with the following phospholipids: PS, phosphatidylserine;PE, phosphatidylethanolamine; PI, phosphatidylinositol: PA, phosphatidicacid; PG, phosphatidylglycerol; PC, phosphatidylcholine: CL,cardiolipin; and SM, sphingomyelin. The specificity profiles of 9D2,1B12, 3G4 and 1B9, as compared to 3SB and D11, are shown in Table 4.TABLE 4 Phospholipid Specificity of Anti-PS Antibodies Name RelativeStrength of Reactivity on ELISA^(1,2) 3SB PS = PA >> CL, PI, PE, PG D11CL = PA >> PS, PI, PE, PG 9D2 PA > PS = CL > PG = PI >> PE 1B12 PS =PA > CL > PE = PI, PG 3G4 PS = PA = PI = PG = CL >> PE 3B10 PS = PA =PI >> PE 1B9 PS only 2G7 PS only 7C5 PS only Annexin V PS = PE = PI =PA > CL > PG

[0987] The 1B9, 2G7 and 7C5 antibodies behave essentially the same.These antibodies recognize only PS and require serum or serum proteinsfor binding to PS. The binding of 1B9, 2G7 and 7C5 to variousphospholipids was assayed only in the presence of 10% bovine serumwhereas binding of the other antibodies was tested either in the absenceor in the presence of serum. For antibodies other than 1B9, 2G7 and 7C5,the presence of serum does not change preference in binding to aparticular phospholipid. This latter group, including 3G4, 3B10 and 9D2,have the preferred property of binding to PS in the absence of serum.

[0988] The 3SB antibody recognizes PS on intact cells in the presenceand absence of serum. The major reactivity of 3SB is with PS, but italso has reactivity with phosphatidic acid, which is a relatively minorcomponent of the plasma membrane (Hinkovska-Galcheva et al., 1989). 3SBis essentially devoid of reactivity with phosphatidylethanolamine andphosphatidylinositol, as well as phosphatidylcholine and sphingomyelin(Table 4).

[0989] PS is the most abundant anionic phospholipid of the plasmamembrane and is tightly segregated to the internal leaflet of the plasmamembrane in normal cells under normal conditions. PS is anaminophospholipid. PE is also an aminophospholipid, but PE is neutral,not anionic. Other than being a neutral aminophospholipid, PE behavessimilarly to PS and is normally tightly segregated to the internalleaflet of the plasma membrane.

[0990] PI is another major anionic phospholipid of the plasma membrane,which is further tightly segregated to the internal leaflet in normalcells under normal conditions. PA and PG are minor anionic phospholipidsof the plasma membrane, which are also normally segregated to theinternal leaflet. CL is an anionic phospholipid present in mitochondrialmembranes, and typically absent from the plasma membrane.

[0991] PC and SM are choline-containing, neutral phospholipids of theplasma membrane. Each of PC and SM are predominantly located on theexternal leaflet under normal conditions.

[0992] In keeping with the inventors' model for differentialaminophospholipid and anionic phospholipid expression between normal andtumor blood vessels, none of the antibodies developed using the selectedprotocol reacted with the neutral phospholipids, PC and SM. The 1B9antibody was specific for PS, whereas 9D2, 1B12 and 3G4 bound to anionicphospholipids and aminophospholipids with the preferences shown in Table4. The 9D2 antibody is also described in Example VI.

EXAMPLE V Externalized Phosphatidylserine is a Global Marker of TumorBlood Vessels

[0993] The present example shows that the exposure of PS occurs onendothelial cells in each of ten different solid tumors growing in miceand is not limited to the L540 tumor model described in Example II.

[0994] Externalized PS in vivo was detected by injecting a monoclonalantibody directed against PS intravenously into mice bearing varioustypes of human or murine tumors. Anti-PS antibodies are shown to bindspecifically to vascular endothelium in all ten different tumor models.Vascular endothelium in normal organs derived from the same mice wereunstained. An isotype-matched control monoclonal antibody did notlocalize to either tumor or normal cells. Apoptotic cells were alsoidentified immunohistochemically, wherein very few endothelial cells intumors expressed markers of apoptosis.

[0995] The present example therefore shows that vascular endothelialcells in tumors but not in normal vessels externalize PS. Most of thetumor endothelial cells having exposed PS were not apoptotic. PS is thusan abundant and accessible marker of tumor vasculature that can be usedfor tumor vessel imaging and therapy.

[0996] A. L540, H358 and HT29 Tumors

[0997] The anti-PS antibody used in these studies was the mousemonoclonal IgM antibody termed 3SB (Example IV, Rote et al., 1993). 3SBmainly binds to PS, but also reacts with PA, a relatively minor anionicphospholipid with a distribution like PS. The anti-CL antibody used wasthe mouse monoclonal IgM antibody termed D11 (Example IV, Rote et al.,1993).

[0998] PS exposure on tumor and normal vascular endothelium was firstexamined in three animal tumor models: L540 human Hodgkin's lymphomas,NCI H358 human non-small cell lung carcinoma (NSCLC) and HT29 humancolorectal carcinomas. To grow the tumors in vivo. 2×10⁶ cells wereinjected into the right flank of SCID mice and tumors allowed to reach0.8-1.2 cm in diameter.

[0999] Mice bearing large tumors (volume above 800 mm³) were injectedintravenously via the tail vein with 20 μg of either anti-PS or anti-CLantibodies. One hour after injection, mice were anesthetized and theirblood circulation was perfused with heparinized saline. Tumors andnormal organs were removed and snap-frozen for preparation ofcryosections. Mouse IgM was detected using goat anti mouse IgM (μspecific)—HRP conjugate followed by development with carbazole. At least10 random fields per section were examined at ×40 magnification and theaverage percentage of positive vessels was calculated.

[1000] The anti-PS antibodies specifically homed to the vasculature ofall three tumors (HT 29. L540 and NCI-H358) in vivo, as indicated bydetection of the mouse IgM. In this first study, the average percentagesof vessels stained in the tumors were 80% for HT 29, 30% for L540 and50% for NCI-H358. Vessels in all regions of the tumors were stained andthere was staining both of small capillaries and larger vessels.

[1001] No vessel staining was observed with anti-PS antibodies in anynormal tissues. In the kidney, tubules were stained in both anti-PS andanti-CL recipients, and this relates to the secretion of IgM throughthis organ. Anti-CL antibodies were not detected in any tumors or normaltissues, except kidney. These findings indicate that only tumorendothelium exposes PS to the outer site of the plasma membrane.

[1002] B. Small and Large L540 Tumors

[1003] To estimate the time at which tumor vasculature loses the abilityto segregate PS to the inner side of the membrane, anti-PS localizationwas examined in L540 tumors ranging in volume from 140 to 1,600 mm³.

[1004] Mice were divided into 3 groups according to their tumor size:140-300, 350-800 and 800-1,600 mm³. Anti-PS Ab was not detected in threemice bearing small L540 tumors (up to 300 mm³). Anti-PS Ab localized in3 animals of 5 in the group of intermediate size L540 tumors and in allmice (4 out of 4) bearing large L540 tumors (Table 5). Percent ofPS-positive blood vessels from total (identified by pan endothelialmarker Meca 32) was 10-20% in the L540 intermediate group and 20-40% inthe group of large L540 tumors (Table 5). TABLE 5 PS ExternalizationDetected in Mid and Large Sized Tumors % PS-Positive Tumor Size (mm³)No. Positive Tumors/Total* Vessels/Total† 350-800 3/5 10-20 850-1,6004/4 20-40

[1005] C. L540, H358, HT29, Colo26, B16 and 3LL Tumors

[1006] Using the same anti-PS (3SB) and anti-CL (D11) antibodies, PSexposure on tumor and normal vascular endothelium was examined infurther studies using an additional three animal tumor models (six intotal): L540 human Hodgkin's lymphomas, NCI H358 human non-small celllung carcinoma (NSCLC), HT29 human colorectal carcinomas, Colo26 mousecolon carcinomas, B 16 mouse melanomas and 3LL mouse lung tumors.

[1007] In these studies, tumors were grown subcutaneously in SCID miceand allowed to reach a volume of 0.4-0.7 cm³. Three or more mice wereused per group. Anti-PS or anti-CL mouse IgM antibodies (30 μg/mouse)were injected intravenously in 200 μl of saline. Thirty minutes later,the mice were sacrificed, exsanguinated and their blood circulationperfused with heparinized saline. Major organs and tumors were harvestedand snap-frozen for preparation of cryosections. Mouse IgM was detectedusing goat anti mouse IgM (μ specific)-HRP conjugate followed bydevelopment with carbazole.

[1008] Serial sections of tumor were stained with a monoclonal antibody,MECA 32, directed against a pan-endothelial marker of mouse vessels.PS-positive vessels were identified morphologically and by theircoincident staining with anti-mouse IgM and MECA 32. At least 10 randomfields per section (0.317 mm²/field) were examined in blinded fashion bytwo independent observers. The percentage of MECA 32-positive vesselsthat stained positively for PS was calculated. Three tumors of each typewere examined in each of two separate studies. The mean values andstandard errors (SE) were calculated. Inter-tumor variation in thenumber of total and PS-positive vessels in each group was approximately10%.

[1009] All six tumors in this study contained PS-positive vessels (Table6). Detection of PS by 3SB was specific since no staining of tumorendothelium was observed with the anti-CL antibody (Table 6; FIG. 1). Novascular localization of anti-PS or anti-CL antibodies was observed innormal organs other than the kidneys (tubule staining in both anti-PSand anti-CL recipients reflects secretion of IgM through this organ).TABLE 6 Specific Localization of Anti-PS Antibodies to Tumor VesselsTissue Anti-PS* Anti-CL L540 tumor 19.3 ± 3.3 0 H358 tumor 15.6 ± 4.1 0HT29 tumor  4.2 ± 1.6 0 B16 tumor 40.6 ± 5.4 0 3LL tumor  5.3 ± 3.7 0Colo 26 tumor 12.4 ± 2.4 0 Adrenal 0 0 Brain 0 0 Heart 0 0 Kidney 0^(♯)0^(†) Intestine 0 0 Liver 0 0 Lung 0 0 Pancreas 0 0 Spleen 0 0 Testis 00

[1010] In these studies, the percentage of PS-positive vessels rangedfrom 10% in Colo 26 tumors to 40% in B16 tumors. Anti-PS IgM was presenton the luminal surface of capillaries and venules in all regions of thetumors. PS-positive vessels appeared to be particularly prevalent in andaround regions of necrosis. Positive vessels usually did not showmorphological abnormalities that were apparent by light microscopy.Occasional vessels located in necrotic areas showed morphological signsof deterioration. Anti-PS antibody (but not anti-CL antibody) alsolocalized to necrotic and apoptotic tumor cells.

[1011] These controlled studies demonstrate that PS is consistentlyexposed on the luminal surface of vascular endothelial in varioustumors, but not in normal tissues, and that the tumor vasculatureexpression is not model-specific.

[1012] D. The Majority of PS-Positive Tumor Vessels are not Apoptotic

[1013] A double labeling technique was used to identify apoptoticendothelial cells in tumor sections. Endothelial cells were identifiedwith the pan-endothelial ceil marker, MECA 32. Apoptotic cells wereidentified immunohistochemically using two independent markers: anactive form of caspase-3, which identifies cytosolic changes in dyingcells (Krajewska et al., 1997), and fragmented DNA, which identifiescells having nuclear alterations (Gavrieli et al. 1992).

[1014] Active caspase-3 was detected by a rabbit anti-caspase-3 specificantibody (R&D. Minneapolis, Minn.) followed by incubation withanti-rabbit IgG conjugated to alkaline phosphatase (AP, Pierce,Rockford, Ill.). Other tumor sections were analyzed by Tunel assay(ApopTag™ kit, Oncor, Md.) using anti-digoxigenin-alkaline phosphataseconjugate as a detecting reagent. Sections were double stained forapoptosis markers (pink) and the endothelial cell marker, MECA 32(brown). Both colors were clearly visible on the same cells, if markersof endothelial cells and apoptotic cells coincided.

[1015] Endothelial cells in five out of six types of tumors (HT29, H358,B16, Colo 26. L540) did not display either of the apoptosis markers(Table 7). The sixth type of tumor. 3LL, displayed a few apoptoticendothelial cells that were located in necrotic areas. In contrast,apoptotic malignant cells were common in all types of tumors. Thepercentage of apoptotic tumor cells ranged from 1-2% in L540 tumors to12.6-19.6% in 3LL tumors. TABLE 7 Expression of Apoptotic Markers inTumors Active caspase-3 Tunel assay Tumor cells Tumor Tumor cells TumorTumor type (% of total)* vessels (% of total) vessels 3LL 19.8 ± 4.3<1.0^(†) 12.6 ± 3.6 0 HT29 13.7 ± 2.3 0  7.8 ± 2.5 0 H358  5.8 ± 2.0 0 4.3 ± 1.6 0 Colo 26  5.3 ± 1.5 0  4.1 ± 1.5 0 B16  4.2 ± 1.8 0  3.5 ±1.6 0 L540  2.3 ± 1.0 0  1.6 ± 0.5 0

[1016] E. MDA-MB-231 and Meth A Tumors

[1017] PS exposure on tumor vascular endothelium was also examined inMDA-MB-231 human breast tumors growing in mice and in mouse Meth Afibrosarcoma growing subcutaneously. The antibody used in these studieswas the 9D2 antibody, generated as described in Example IV, which isreactive with anionic phospholipids.

[1018] As described in detail in Example VI, 9D2 localized to tumorvessels in L540. NCI-H358 and B16 tumors, as well as in models ofMDA-MB-231 breast tumor growing orthotopically in the mammary fat padsof SCID mice and mouse Meth A fibrosarcoma growing subcutaneously. 9D2localized to tumor vessels in all of five tumors. Vascular endotheliumin the tumors showed distinct membrane staining. 9D2 antibody alsolocalized to the membrane and cytosol of necrotic and apoptotic tumorcells. No vascular localization of 9D2 antibody was observed in 9 of the10 normal organs that were examined, with non-specific staining of thetubules in the kidney being observed.

[1019] Double-staining studies were also performed in which mice bearingorthotopic MDA-MB-231 breast tumors were injected i.v. with biotinylated9D2 antibody and frozen sections later stained with FITC-conjugatedMECA32 (Example VI). About 40% of MECA 32-positive vessels bound 9D2.

[1020] F. MD-MBA-435 Tumors

[1021] In a further breast cancer model, PS exposure on tumor vascularendothelium was examined in MDA-MB-435 human breast cancer cells growingin mice. The antibody used in these studies is a chimeric version of the3G4 antibody (ch3G4). The 3G4 antibody generation is described inExample IV, and the production of the chimeric 3G4 antibody is detailedin Example XIX. The localization of ch3G4 to tumor vascular endotheliumin the MDA-MB-435 model is described in more detail in Example XIX andshown in FIG. 22.

[1022] Briefly, tumors were established using MD-MBA-435s cells andbiotinylated versions of the chimeric 3G4 antibody and a control IgG ofirrelevant specificity were administered. Tumor sections were stainedwith Cy3-conjugated streptavidin to detect the biotinylated proteins.Double staining with the MECA 32 antibody followed by FITC-taggedanti-rat IgG secondary antibody was conducted to detect vascularendothelium. This detection method labeled the biotinylated proteins andthe vascular endothelium using red and green, so that biotinylatedproteins bound to the endothelium appear yellow in a converged image(FIG. 22). This study showed specific localization of the chimeric 3G4antibody to tumor vascular endothelium.

[1023] G. RIP-Tag Tumors

[1024] For the tenth model, PS exposure on tumor vascular endotheliumwas examined in a “RIP-Tag” transgenic mouse model (RIP1-Tag 2) ofmultistage carcinogenesis. In this transgenic mouse model, every mousedevelops islet tumors of the pancreas by 12-14 weeks of age as a resultof expression of the SV40 T antigen (Tag) oncogene in insulin-producingbeta-cells. Tumors develop in multiple stages from hyper-proliferativeislets, and require an angiogenic switch in order to progress towardsmalignancy. Matrix metalloprotinase-9 controls the angiogenic switch(REF).

[1025] 9D2 localization studies were conducted in the RIP1-Tag2 model incollaboration with Dr. Donald McDonald, Professor of Pathology at UCSF.9D2 was injected intravenously into RIP1-Tag2 mice starting at 10 weeksof age, when all mice have small, highly vascularized, solid tumors.Double staining of thick (80 μm) tumor sections was performed toidentify localized 9D2 and CD31 in tumors and normal pancreas.Approximately 50% of vessels (CD31 positive) in pancreatic tumors hadlocalized 9D2, whereas vessels in normal islets were unstained. Miceinjected with control rat IgM had weak and infrequent staining of tumorvessels. Some leakage of 9D2 and control rat IgM into extravasculartissues beyond the endothelium was also apparent.

[1026] The present example therefore confirms that vascular endothelialcells in tumors externalize PS and anionic phospholipids to theirluminal surface, where they can be bound by anti-PS antibodies in vivo.PS is absent from the external surface of vascular endothelial cells innormal tissues, indicating that PS-recognizing antibodies, annexin V andother ligands can be used for delivering cytotoxic drugs, coagulants andradionuclides for the selective imaging or destruction of vessels insolid tumors.

[1027] PS-positive tumor endothelium appeared, for the most part, to beviable in the tumors used in this study. It does not display markers ofapoptosis, it is morphologically intact and metabolically active, asindicated by its expression of VCAM-1, E-selectin and other rapidlyturned-over proteins. Although often regarded as an indicator ofapoptosis, PS exposure has been observed in several types of viablecells, including malignant cells (Rao et al., 1992), (Utsugi et al,1991) activated platelets (Rote et al., 1993), and embryonictrophoblasts at various stages of migration, matrix invasion and fusion(Adler et al., 1995).

[1028] Lack of correlation between PS exposure and commitment to celldeath has been also shown on pre-apoptotic B lymphoma cells that restorePS asymmetry and grow normally after removal of the pro-apoptoticstimulus (Hammill et al., 1999). In normal viable cells, PS exposure isprobably triggered by surface events, such as ligand-receptorinteractions, that induce Ca²⁺ fluxes into the cells (Dillon et al.,2000). Ca²⁺ fluxes activate scramblase (Zhao et al., 1998) andsimultaneously inhibit aminophospholipid translocase (Comfurius et al.1990).

[1029] PS on tumor vessels is attractive as a target for cancer imagingor therapy for several reasons: it is abundant (approximately 3×10⁶molecules per cell); it is on the luminal surface of tumor endothelium,which is directly accessible for binding by vascular targeting agents inthe blood; it is present on a high percentage of tumor endothelial cellsin diverse solid tumors, and it is absent from endothelium in all normaltissues examined to date. Unconjugated antibodies, vascular targetingagents and imaging agents directed against PS or tumor vasculature cantherefore be used for the detection and treatment of cancer in man.

EXAMPLE VI Anionic Phospholipids are Exposed on the Surface of TumorBlood Vessels

[1030] Anionic phospholipids are largely absent from the externalleaflet of the plasma membrane of mammalian cells under normalconditions. Exposure of phosphatidylserine, for example, on the cellsurface occurs during apoptosis, necrosis, cell injury, cell activationand malignant transformation. The present example shows that anionicphospholipids are upregulated on tumor vasculature in vivo, asdemonstrated by localization of both a specific antibody and a naturalligand that binds to anionic phospholipids.

[1031] A monoclonal antibody, 9D2, which specifically recognizes anionicphospholipids, was injected into mice bearing a variety of orthotopic orectopic tumors. Other mice received annexin V, a natural ligand thatbinds to anionic phospholipids. Both 9D2 and annexin V specificallylocalized to vascular endothelium in all tumors and also to tumor cellsin and around regions of necrosis. Between 15 and 40% of endothelialcells in tumor vessels were stained. No localization was detected onnormal endothelium.

[1032] Various factors and tumor-associated conditions known to bepresent in the tumor microenvironment were examined for their ability tocause exposure of anionic phospholipids in cultured endothelial cells,as judged by 9D2 and annexin V binding. Hypoxia/reoxygenation, acidity,thrombin and inflammatory cytokines all induced exposure of anionicphospholipids. Hydrogen peroxide was also a strong inducer. Combinedtreatment with inflammatory cytokines and hypoxia/reoxygenation hadgreater than additive effects. The demonstrated exposure of anionicphospholipids on tumor endothelium in vivo is thus likely to be causedby injury and activation by cytokines and reactive oxygen species.Irrespective of the mechanism, anionic phospholipids are markers oftumor vessels that can now be used for tumor vessel targeting, imagingand therapy.

[1033] A. Materials and Methods

[1034] 1. Materials

[1035] Na¹²⁵I was obtained from Amersham (Arlington Heights, Ill.).Dulbecco's modified Eagle's tissue culture medium and Dulbecco PBScontaining Ca²⁺ and Mg²⁺ were obtained from Gibco (Grand Island, N.Y.).Fetal calf serum was obtained from Hyclone (Logan, Utah).L-α-phosphatidylserine, L-α-phosphatidylcholine, cardiolipin,L-α-phosphatidylethanolamine, L-α-phosphatidylinositol, sphingomyelin,phosphatidic acid, phosphatidylglycerol, O-phenylenediamine, hydrogenperoxide and thrombin were from Sigma (St. Louis, Mo.). Flat bottomplates with 24 wells were obtained from Falcon (Becton Dickinson andCo., Lincoln Park, N.J.).

[1036] Recombinant hepatocyte growth factor (HGF or scatter factor) andactinomycin D was from Calbiochem (San Diego, Calif.). Recombinantmurine interleukin-1 alpha, beta and tumor necrosis factor alpha (TNF α)were purchased from R&D Systems (Minneapolis, Minn.). Interferon ofUniversal Type I (hybrid protein that substitutes for all types ofinterferons) was purchased from PBL Biomedical Laboratories (NewBrunswick, N.J.). Recombinant human vascular endothelial growth factor121 (VEGF), human platelet-derived growth factor-BB, interleukin-6(IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and humanfibroblast growth factor-2 (FGF-2) were purchased from PeproTech (RockyHill, N.J.).

[1037] 2. Antibodies

[1038] MECA 32, a pan mouse endothelial cell antibody, was obtained fromDr. E. Butcher (Stanford University, CA) and served as a positivecontrol for immunohistochemical studies. Details of this antibody havebeen published (Leppink et al., 1989). Rabbit anti-rat immunoglobulin,rat-anti mouse immunoglobulin and goat-anti mouse and anti-rat secondaryantibodies conjugated to horseradish peroxidase (HRP) were purchasedeither from Daco (Carpinteria, Calif.) or from Jackson ImmunoresearchLabs (West Grove, Pa.).

[1039] The 9D2 antibody used in these studies was generated as describedin Example IV. 9D2 is a rat monoclonal antibody reactive with anionicphospholipids. Further characterization of the phospholipid specificityof 9D2 is given in the results section of this example.

[1040] 3. Cells

[1041] L540Cy Hodgkin lymphoma cells, derived from a patient withend-stage disease, were provided by Prof. V. Diehl (Köln, Germany).NCI-H358 human non-small cell lung carcinoma was provided by Dr. AdiGazdar (Southwestern Medical Center, Dallas, Tex.). Meth A mousefibrosarcoma and MDA-MB-231 human breast carcinoma were obtained fromAmerican Type Cell Collection (Rockville, Md.). The mouse brainendothelioma line, bEnd.3, was provided by Prof. Werner Risau (Max PlankInstitution, Munich, Germany) and was maintained in DMEM with 10% FBS.Adult bovine aortic endothelial (ABAE) cells were purchased fromClonetics (San Diego, Calif.; Walkerville, Md.). ABAE cells weremaintained in DMEM with 10% serum and 2 ng/ml of bFGF.

[1042] 4. Tissue Culture

[1043] bEnd.3, ABAE cells and all tumor cells except L540Cy lymphomawere maintained in DMEM supplemented with 10% fetal calf serum, 2 mML-glutamine. 2 units/ml penicillin G and 2 μg/ml streptomycin. L540Cycells were maintained in RPMI 1640 containing the same additives. Cellswere sub-cultured once a wk. Trypsinization of bEnd.3 cells wasperformed using 0.125% trypsin in PBS containing 0.2% EDTA. For in vitrostudies, endothelial cells were seeded at a density of 10×10³ cells/mlin 1 ml of culture medium in 24 well plates and incubated 48-96 h beforebeing used in the assays. Medium was refreshed 24 h before each study.

[1044] 5. Reactivity with Plastic-Immobilized Phospholipids

[1045] Phospholipids were dissolved in n-hexane to a concentration of 50μg/ml. 100 μl of this solution was added to wells of 96-well microtiterplates. After evaporation of the solvent in air, the plates were blockedfor 2 h with 10% fetal bovine serum diluted in DPBS containing 2 mM Ca²⁺(binding buffer).

[1046] 9D2 antibody or annexin V were diluted in the binding buffer inthe presence of 10% serum at an initial concentration of 6.7 nM. Serialtwo-fold dilutions were prepared in the plates (100 μl per well). Theplates were then incubated for 2 h at room temperature. The plates werewashed and the 9D2 and annexin V were detected by goat anti-rat IgMconjugated to HRP and rabbit anti-human annexin V followed by goatanti-rabbit IgG conjugated to HRP (all diluted 1:1000), respectively.Secondary reagents were detected by using chromogenic substrate OPDfollowed by reading plates at 490 nm using a microplate reader(Molecular Devices, Palo Alto, Calif.).

[1047] The specificity of the 9D2 antibody binding was validated byusing control rat IgM of irrelevant specificity (Pharmingen, San Diego,Calif. The specificity of annexin V binding to phospholipids, which isCa²⁺-dependent, was determined by diluting the reagent in the DPBScontaining 5 mM EDTA. Additional negative controls consisted of washingthe plates with the binding buffer containing 0.2% of a detergent Tween20. This treatment extracts lipids, thus removing the phospholipid thatwas absorbed to plastic. Neither 9D2 antibody nor annexin V bound todetergent-washed plates.

[1048] 6. Detection of Anionic Phospholipids on the Surface of CulturedEndothelial Cells

[1049] Endothelial cells were grown until they reached approximately 70%confluence. To induce PS exposure, cells were treated with H₂O₂ (200 μM)for 1 h at 37° C. Control and treated slides were washed with DPBScontaining Ca²⁺ and Mg²⁺ and fixed with 0.25% of glutaraldehyde dilutedin the same buffer. Excess aldehyde groups were quenched by incubationwith 50 mM of NH₄Cl for 5 min. To examine the effect of detergents andorganic solvents on detection of phospholipids, some slides werepre-incubated with acetone (5 min) or with PBS containing 1% (v/v)Triton™ X-100.

[1050] Cells were washed with DPBS (containing Ca²⁺, Mg²⁺ and 0.2% (w/v)gelatin) and incubated with 1 μg/ml of biotinylated annexin V(Pharmingen, San Diego, Calif.) or with 1 μg/ml of 9D2 antibody. After 2h of incubation, cells were washed with 0.2% gelatin buffer and wereincubated with streptavidin-HRP (1:500 dilution). Rat IgM of irrelevantspecificity and streptavidin alone were used as negative controls inthese studies. All steps were performed at room temperature. HRPactivity was measured by adding O-phenylenediamine (0.5 mg/ml) andhydrogen peroxide (0.03% w/v) in citrate-phosphate buffer, pH 5.5. After15 min, 100 μl of supernatant were transferred to 96 well plates, 100 μlof 0.18 M H₂SO₄ were added and the absorbance was measured at 490 nm.Alternatively, PS-positive cells were detected by addition of carbazolesubstrate, resulting in insoluble red-brownish precipitate. Each studywas performed in duplicate and repeated at least twice.

[1051] 7. Inhibition of 9D2 and Annexin V Binding to Phospholipids byLiposomes

[1052] The specificity of phospholipid recognition was further confirmedby competition assays with various liposomes. Liposomes were preparedfrom solutions of 5 mg of a single phospholipid in chloroform. Thesolutions were dried under nitrogen to form a thin layer in around-bottomed glass flask. Ten ml of Tris buffer (0.1 M, pH 7.4) werethen added and the flask was sonicated five times for 2 min. 9D2 orannexin V (6.66 nM) were pre-incubated with 200 μg/ml of liposomalsolution for 1 h at room temperature. The mixture was added tophospholipid-coated plates or endothelial cell monolayers. The abilityof 9D2 to bind to an immobilized phospholipid or cell surface in thepresence or absence of the different liposomes was determined asdescribed above.

[1053] 8. Competition of 9D2 and Annexin V for Binding to Immobilized PS

[1054] Biotinylated 9D2 antibody and annexin V were prepared byincubating purified proteins with a 10-fold molar excess ofN-hydroxysuccinimide biotin (Sigma, Mo.) for 1 h at room temperature.Free biotin was removed by dialysis against PBS. The biotinylationprocedure did not impair the PS-binding capacity of either protein. Forcompetition studies, unmodified and biotinylated proteins were premixedwith a 10-fold molar excess of unmodified proteins. The mixtures werethen added to PS-coated plates. Bound reagents were detected bystreptavidin-HRP conjugate diluted 1:1000. The binding to PS of eachreagent in the absence of a competitor was taken as the 100% value.

[1055] 9. Growth of Subcutaneously Implanted Tumors

[1056] For localization studies. 2×10⁷ L540 human Hodgkin's lymphomacells or 1×10⁷ cells of other tumor types were injected subcutaneouslyinto the right flank of SCID mice (Charles River. Wilmington, Mass.).Tumors were allowed to reach a volume of 0.4-0.7 cm³. A minimum of threeanimals per group was used. Studies were replicated at least threetimes.

[1057] 10. Orthotopic Model of Human MDA-MB-231 Breast Carcinoma

[1058] Female nu/nu or SCID mice were purchased from Charles River.MDA-MB-231 human mammary carcinoma cells were implanted into the mammaryfat pad according to a published protocol (Price, 1996). Briefly, micewere anesthetized and a 5-mm incision was made in the skin over thelateral thorax. The mammary pad was exposed to ensure the correct sitefor injection of 1×10⁴ MDA-MB-231 cells re-suspended in 0.1 ml ofsaline.

[1059] 11. Detection of Anionic Phospholipids in Tumor Bearing Mice InVivo

[1060] Immunohistochemical techniques, in which 9D2 or annexin V areapplied directly to sections of frozen tissues, do not discriminatebetween anionic phospholipids on the inner leaflet and the outer leafletof the plasma membrane. To detect externally-positioned phospholipids,methods were performed essentially as previously described (Example V;Ran et al., 1998). Tumor-bearing SCID mice were injected intravenouslywith either 50 μg of 9D2 or biotinylated 9D2 antibody or 100 μg ofbiotinylated annexin V. Sixty min later mice were sacrificed and theirblood circulation was exsanguinated and perfused with heparinized salineas previously described (Burrows et al., 1992). All major organs andtumor were harvested and snap-frozen for preparation of cryosections.

[1061] Sections were blocked with PBS containing 10% serum. To preventloss of phospholipids during slide processing, detergents and organicsolvents were omitted from blocking and washing buffers. Rat IgM wasdetected using goat anti rat IgM (μspecific)-HRP conjugate followed bydevelopment with carbazole or DAB (Fries et al., 1993). Biotinylatedreagents were detected by streptavidin conjugated to HRP.

[1062] Tumor sections derived from mice injected with saline or rat IgMof irrelevant specificity served as negative controls. Additionalcontrols consisted of incubating the slides in 1% Triton solution or inacetone for 10 min. These treatments extract phospholipids. No signalwas detected under these conditions. The number of positive vessels perhigh power field was determined at magnification of ×100. At least 10fields per section were examined and the average percentage of positivevessels was calculated. Staining of the sections by this method for thepresence of 9D2 or annexin V detects cells having externalized anionicphospholipids that were accessible for binding by the reagents in vivo.

[1063] 12. Identification and Quantification f PS-Positive Tumor Vessels

[1064] Structures with localized 9D2 antibody or annexin V wereidentified as blood vessels by morphological appearance on DAB-stainedsections and by co-incident staining with the pan-endothelial cellmarker, MECA 32 on serial sections of frozen tissues. Quantification onDAB-stained sections was done by counting vessels stained by MECA 32,9D2 or annexin V in serial sections of a tumor. Six slides of each tumortype derived from 6 mice injected with 9D2 antibody, control rat IgM orannexin V were examined. At least 10 random fields per section (0.317mm²/field) were scored in blinded fashion by two independent observers.The mean numbers and standard errors of vessels stained by 9D2, annexinV or MECA 32 were calculated. The mean number of 9D2 or annexinV-positive vessels determined in each tumor type group was compared tothe mean number of MECA 32-positive vessels in the same tumor group. Thepercentage of 9D2 or annexin V-positive vessels was calculated.

[1065] In further studies, mice bearing MDA-MB-231 tumors (0.3-0.7 cm³in volume) were injected intravenously with 50 μg of biotinylated 9D2,control IgM or annexin V (six mice per group). Biotinylated reagentswere first incubated with streptavidin-Cy3 conjugate, washed in PBS,then incubated with MECA 32 antibody followed by FITC-tagged anti-ratIgG secondary antibody. Single images, taken with appropriate filtersfor Cy3 (red) and FITC (green) fluorescence respectively, were capturedby digital camera and transferred to a computer. Images of 10 randomfields (0.317 mm²/field) demonstrating yellow color (a product of mergedgreen and red fluorescence) were superimposed with the aid of Metaviewsoftware. The same method was used to analyze tumors from mice injectedwith control rat IgM or saline. The percentage of vessels with localized9D2 or annexin V was calculated as follows: mean number of yellowvessels per field divided by mean number of green (total) vesselsmultiplied by 100.

[1066] B. Results

[1067] 1. Phospholipid Specificity of 9D2 Antibody and Annexin V

[1068] The 9D2 antibody specifically recognized anionic phospholipids(PS, PA, CL, PI, PG) and had no significant reactivity with neutralphospholipids (PE, PC and SM) in ELISA (FIG. 2A; Table 8). The order ofstrength of binding of 9D2 to phospholipids in ELISA was PA>PS=CL>PG=PI.The binding was antigen-specific since no binding was observed withseveral control rat IgM of irrelevant specificity. Binding of 9D2 to anyof the anionic phospholipids adsorbed to ELISA plates was blocked byliposomes prepared from any of the anionic phospholipids, but not byliposomes prepared from any of the neutral phospholipids. TABLE 8Phospholipid Specificity of 9D2 and Annexin V Abundance and location inthe plasma membrane Phospholipid under normal EC₅₀ of binding (pM) NameType conditions^(a) 9D2 Annexin V PS Anionic Major PL (15%), located 12100 amino-PL on inner side PA Anionic PL Minor PL (less than 1%) 2 100PG Anionic PL Minor PL (less than 1%) 100 250 PI Anionic PL Major PL(7%), mainly 100 50 located on the inner side CL Anionic PL Absent fromthe plasma 15 130 membrane PE Neutral Major PL (22%), mainly >8000 100amino-PL located on inner side SM Neutral Major PL (9%), locatedon >8000 >8000 choline-PL the outer side PC Neutral Major PL (46%),located on >8000 >8000 choline-PL the outer side

[1069] Annexin V also bound to anionic phospholipids, but its bindingwas less specific than that of 9D2 in that it also bound strongly to theneutral phospholipid, PE. The order of strength of binding of annexin Vto phospholipids in ELISA was PI>PS=PE=PA=CL>PG (Table 8). Thesefindings for annexin V are consistent with earlier data (Andree et al.,1990).

[1070] The binding of 9D2 was unaffected by the presence of 5 mM EDfA,showing it did not require Ca²⁺ for binding to anionic phospholipids. Incontrast, the binding of annexin V to anionic phospholipids wasabolished in the presence of 5 mM EDTA, as expected from its knowndependence on Ca²⁺ for binding to anionic phospholipids or PE(Schlaepfer et al., 1987; Blackwood and Ernst, 1990).

[1071] Neither 9D2 nor annexin V bound to ELISA plates that had beencoated with phospholipids but then washed with 0.2% Tween in saline,confirming that their binding was to the absorbed phospholipids. 9D2 andannexin V did not bind detectably to heparin, heparan sulfate or todouble or single stranded DNA.

[1072] 2. 9D2 Antibody and Annexin V Do Not Cross-Block Each Other'sBinding to PS

[1073] To examine whether 9D2 antibody and annexin V compete for bindingto PS, cross-blocking studies were performed using biotinylated proteinson PS-coated plates. Binding of biotinylated 9D2 antibody and annexin Vwas blocked by a 10-fold molar excess of unmodified 9D2 and annexin V,respectively (Table 9). However, unmodified annexin V did not affect theability of biotinylated 9D2 to bind to the PS plate. Likewise, additionof unmodified 9D2 antibody did not alter the ability of biotinylatedannexin V to bind to the PS plate (Table 9). TABLE 9 9D2 and Annexin VDo Not Cross-Block Binding to PS PS-binding protein Competitor^(a)Binding (% Control)^(b) Biotinylated annexin V Annexin V 8% Biotinylated9D2 Annexin V 93% Biotinylated annexin V 9D2 95% Biotinylated 9D2 9D2 5%

[1074] These results indicate that 9D2 antibody and annexin V do notcross-block each other binding to PS-coated plates, either because theyrecognize different epitopes on the PS molecule or differentconformations of PS adsorbed on plastic.

[1075] 3. Binding to Externalized Anionic Phospholipids on Cell Surfaces

[1076] The binding of 9D2 antibody and annexin V to cell surfaces wasexamined using mouse bEnd.3 endothelioma cells or bovine ABAE cells.Neither 9D2 nor annexin V bound to non-permeabilized monolayers ofeither cell type under quiescent conditions. This indicates that themajority of anionic phospholipids of the plasma membrane are normallysequestered to the cytosolic domain. In contrast, strong staining wasobserved when cells were pre-incubated with TNFα and actinomycin D underconditions that caused apoptosis in 90-100% of the endothelial cells.

[1077] To confirm that 9D2 and annexin V were binding to phospholipidson cell surfaces. H₂O₂-treated bEnd.3 cells were incubated with 9D2antibody or annexin V in the presence or absence of various competingliposomes. Anionic phospholipids become exposed on non-apoptotic, viablebEnd.3 cells when they are pre-treated with a sub-toxic concentration(100-200 μM) of H₂O₂ (Ran et al., 2002).

[1078] The binding of 9D2 antibody to H₂O₂-treated bend.3 cells wasinhibited by liposomes containing anionic phospholipids but not byliposomes containing neutral phospholipids (FIG. 3). The magnitude ofinhibition of 9D2 binding to cells varied in the order PA>PS>CL>PG>PI,in close agreement with the results obtained using plastic-immobilizedphospholipids (FIG. 2A and FIG. 2B). Similarly, the binding of annexin Vto H₂O₂-treated cells was blocked by liposomes containing PS, PA, PE, CLand, to a lesser extent, PI and PG. Liposomes containing SM or PC didnot block annexin V binding to cells, all in agreement with the resultsobtained using plastic-immobilized phospholipids.

[1079] These results confirm that 9D2 binds to anionic phospholipids inthe H₂O₂-treated endothelial cells, whereas annexin V binds to PE inaddition of anionic phospholipids.

[1080] 4. Detection of Externalized Anionic Phospholipids on Cells InVivo

[1081] Direct immunohistochemical techniques, in which 9D2 or annexin Vare applied directly to sections of frozen tissues, do not discriminatebetween anionic phospholipids on the inner leaflet and the outer leafletof the plasma membrane. To detect externally-positioned phospholipids,9D2 and annexin V were injected intravenously into tumor-bearing miceand localization to tumor vessels was determined by indirectimmunohistochemistry.

[1082] Mice bearing various types of solid tumors were injectedintravenously with 9D2 antibody or biotinylated annexin V, and one hourlater, were exsanguinated and the tumors and normal tissues were removedand frozen sections were prepared. Frozen sections of tissues were cutand stained with HRP-labeled anti-rat IgM or with HRP-labeledstreptavidin to determine to which cells the 9D2 and annexin V had boundafter injection. Blood vessels were identified morphologically, and fromtheir positive staining by the pan-endothelial cell antibody, MECA 32,on serial sections.

[1083] 5. Biodistribution of 9D2 Antibody and Annexin V in Tumor BearingMice

[1084] 9D2 antibody and annexin V localized to tumor vessels in all offive tumors included in this study (FIG. 4; Table 10). The tumors were:human MDA-MB-231 breast tumor growing orthotopically in the mammary fatpads of SCID mice; human L540 Hodgkin's tumor growing subcutaneously;human NCI-H358 NSCLC growing subcutaneously; mouse B16 melanoma growingsubcutaneously and mouse Meth A fibrosarcoma growing subcutaneously.TABLE 10 Specific Localization of 9D2 and Annexin V to Tumor VesselsTissue 9D2 Antibody^(a) Rat IgM control Annexin V^(b) Tumors MDA-MB-23140.6 ± 5.4 — 45.3 ± 5.6 L540cy 19.3 ± 3.3 — 16.7 ± 3.9 NCI-H358 15.6 ±4.1 — ND B16 23.4 ± 4.5 — 21.3 ± 6.6 Meth A 25.7 ± 6.8 — ND NormalAdrenal — — — Brain — — — Heart — — — Kidney —^(c) —^(c) — Intestine — —— Liver — — — Lung — — — Pancreas — — — Spleen — — — Testis — — — #typewere analyzed. The mean number of MECA 32-positive vessels per 0.317 mm²field was 23, 25, 21, 18 and 19 ± 10 vessels for MDA-MB-231, L540cy,H358, B16 and Meth A tumors, respectively

[1085] 9D2 and annexin V gave essentially the same patterns of staining.Localization of the 9D2 antibody to tumor vessels was specific since nostaining of tumor endothelium was observed with rat IgM of irrelevantspecificity. Presumably, leakage of the control rat IgM out of tumorvessels occurred to some extent, but the staining of extravascular IgMwas too diffuse or too weak to discern by indirect immunohistochemistry.

[1086] No vascular localization of 9D2 antibody or annexin V wasobserved in nine of the ten normal organs that were examined (Table 10).In the kidney, staining of tubules was observed that appeared not to beantigen specific. Tubules were stained in both 9D2 and control rat IgMrecipients, presumably because of secretion of IgM or its metabolitesthrough this organ. The ovaries, a site of physiological angiogenesis,were not examined.

[1087] The percentage of 9D2 and annexin V positive vessels ranged from40% in MDA-MB-231 tumors to 15% in H358 tumors. Anionicphospholipid-positive vessels were present on the luminal surface ofcapillaries and vessels in all regions of the tumors, but wereparticularly prevalent in and around regions of necrosis. Most anionicphospholipid-positive vessels did not show morphological abnormalitiesthat were apparent by light microscopy. Occasional vessels, particularlythose located in necrotic areas, showed morphological signs ofdeterioration. 9D2 antibody and annexin V also localized to necrotic andapoptotic tumor cells whereas localization of the control IgM was notdetectable (FIG. 4).

[1088] These findings demonstrate that anionic phospholipids are presenton the luminal surface of vascular endothelial cells in various tumorsbut not in normal tissues.

[1089] 6. Double Staining Studies

[1090] Double staining studies were also performed in which mice bearingorthotopic MDA-MB-231 breast tumors were injected intravenously withbiotinylated 9D2 antibody, biotinylated control IgM or biotinylatedannexin V. One hour later, the mice were exsanguinated, and their tumorswere removed and frozen sections were cut. The tumor sections were thenstained with Cy3-conjugated streptavidin to detect the biotinylatedproteins and with FITC-conjugated MECA32 to detect vascular endothelium.This detection method labeled the biotinylated proteins and the vascularendothelium by red and green. Where the biotinylated proteins are boundto the endothelium, the converged image appears yellow.

[1091] In these studies, the biotinylated 9D2 and annexin V appearedmostly to be bound to the vascular endothelium, because their stainingpatterns converged with that of MECA 32. About 40% of MECA 32 positivevessels bound 9D2 and annexin V, in close agreement with the resultsobtained by indirect immunohistochemistry. However, leakage of thebiotinylated proteins into the tumor interstitium was detected by doublestaining, whereas it was not apparent by indirect immunohistochemistry.

[1092] Biotinylated proteins were visible outside the vascularendothelium around a minority (about 5%) of vessels. In tumors from micethat had been injected with biotinylated rat IgM of irrelevantspecificity, the biotinylated IgM had also leaked into the tumorinterstitium around a similar percentage (about 5%) of vessels, butmostly appeared not to be bound by the vascular endothelium. Presumably,the detection of extravasated 9D2 and annexin V by the double stainingtechnique, but not by the indirect immunohistochemistry technique,reflects the greater sensitivity of the former technique and the greaterprecision with which two staining patterns can be compared. Non-injectedcontrol tumors were completely unstained by streptavidin-Cy3, indicatingthat red fluorescence corresponds to a localized protein.

EXAMPLE VII Anionic Phospholipid Membrane Translocation in a TumorEnvironment

[1093] The discovery of aminophospholipids and anionic phospholipids asin vivo surface markers unique to tumor vascular endothelial cellsprompted the inventors to further investigate the effect of a tumormicroenvironment on the translocation and outer membrane expression ofsuch molecules. The present example shows that exposing endothelialcells in vitro to certain conditions that mimic those in a tumorduplicates the earlier observed aminophospholipid and anionicphospholipid surface expression in intact, viable cells.

[1094] A. Materials and Methods

[1095] 1. Iodination of Annexin V

[1096] Recombinant human annexin V was purified from E. coli transformedwith ET12a-Panionic phospholipid1 plasmid (obtained from Dr. J. Tait,University of Washington, Seattle). The purity of the protein and thebinding to PS were confirmed on SDS-PAGE and on PS-coated plastic,respectively. Rabbit polyclonal, affinity-purified anti-annexin Vantibodies were used to detect annexin V bound to PS. Annexin V wasradiolabeled with ¹²⁵I using Chloramine T as described by Bocci (1964).The specific activity was approximately 1×10⁶ cpm per μg of protein, asmeasured by a Bradford assay (1976).

[1097] 2. Endothelial Cell Treatment

[1098] Endothelial cells were treated with cytokines or growth factorsat the concentrations listed in Table 11. All reagents were diluted inmedium containing 10% serum and incubated with the cells at 37° C. for24 h.

[1099] To study the effect of hypoxia, cells were seeded on 24 wellplates and were incubated in a humidified normoxic atmosphere (21% O₂,5% CO₂) for 48 h before being transferred to a humidified hypoxicatmosphere (1% O₂, 5% CO₂, 94% N₂) in a sealed chamber (BillupsRothenberg Inc., Del Mar, Ca). Cells were incubated in a hypoxic chamberfor 24 h at 37° C. and were then returned to a normoxic environment for4 h at 37° C. The cells were compared to a parallel culture from anidentical passage, seeded on the same day and maintained entirely undernormoxic conditions. In some studies, IL-1α (10 ng/ml) and TNFα (20ng/ml) were added to the medium before transfer to the hypoxic chamber.

[1100] To examine the effect of an acidic microenvironment, cells wereexposed to the growth medium lacking bicarbonate, which was adjusted todifferent pHs (ranging between 7.3 and 6.2) with the required amount ofHCl. Cells were incubated at 37° C. in the absence of CO₂. It wasconfirmed that culture media held the assigned pH during the 24 h periodof culture. These experimental conditions were not toxic to eitherbovine or mouse endothelial cells and had no effect on cell morphologyor viability of the attached monolayer.

[1101] 3. Detection of PS on Cultured Endothelial Cells by ¹²⁵I-LabeledAnnexin V

[1102] After treatment with the reagents described above, treated andcontrol cells were incubated with 7.1 pmoles of ¹²⁵I-labeled annexin V(200 μl/well) in the binding buffer. After 2 h incubation at roomtemperature, cells were washed extensively and dissolved in 0.5 M ofNaOH. The entire volume of 0.5 ml was transferred to plastic tubes andcounted in a gamma counter. Non-specific binding was determined in thepresence of 5 mM EDTA and was subtracted from experimental values. Theresults were expressed as net pmoles of cell-bound annexin V, normalizedper 1×10⁶ cells.

[1103] Maximal binding of annexin V was determined on cellssimultaneously treated with actinomycin D and TNFα (50 ng/ml of eachcomponent). As has been previously reported, these agents causeapoptosis and PS exposure in 90-100% of endothelial cells (Lucas et al.,1998). Basal binding of ¹²⁵I-annexin V to untreated cells was determinedin the presence of medium with 10% serum. The amount of ¹²⁵I-annexin Vthat bound to the untreated cultures was subtracted from that in thetreated cultures. Exposure of PS was calculated according to thefollowing formula: cell-bound annexin V (pmoles) under experimentalconditions divided by maximal annexin V binding (pmoles), multiplied by100. Each study was performed in duplicate and was performed at leastthree times. Mean values were calculated. The SE of the mean values fromthree separate experiments was less than 5%.

[1104] B. Results

[1105] 1. Induction by H₂O₂

[1106] Mouse bEnd.3 endothelial cells were seeded at an initial densityof 50,000 cells/well. Twenty-fours later cells were incubated withincreasing concentrations of H1202 (from 10 μM to 500 μM) for 1 hour at37° C. or left untreated. At the end of the incubation, cells werewashed 3 times with PBS containing 0.2% gelatin and fixed with 0.25%glutaraldehyde. Identical wells were either stained with anti-PS IgM ortrypsinized and evaluated for viability by the Trypan Blue exclusiontest. For the anti-PS staining, after blocking with 2% gelatin for 10min., cells were incubated with 2 μg/ml of anti-PS antibody, followed bydetection with anti-mouse IgM-HRP conjugate.

[1107] Exposing endothelial cells to H₂O₂ at high concentrations causesPS translocation in ˜90% cells. However, this is accompanied bydetachment of the cells from the substrate and cell viability decreasingto about 50-60%. The association of surface PS expression withdecreasing cell viability is understandable, although it is stillinteresting to note that ˜90% PS translocation is observed with only a50-60% decrease in cell viability.

[1108] Using lower concentrations of H₂O₂ resulted in significant PSexpression without any appreciable reduction in cell viability. Forexample, PS was detected at the cell surface of about 50% of cells inall H₂O₂ treated wells using H₂O₂ at concentrations as low as 20 μM. Itis important to note that, under these low H₂O₂ concentrations, thecells remained firmly attached to the plastic and to each other, showedno morphological changes and had no signs of cytotoxicity. Detailedanalyses revealed essentially 100% cell-cell contact, retention ofproper cell shape and an intact cytoskeleton.

[1109] The 50% PS surface expression induced by low levels of H₂O₂ wasthus observed in cell populations in which cell viability was identicalto the control, untreated cells (i.e., 95%). The PS expressionassociated with high H₂O₂ concentrations was accompanied by cell damage,and the PS-positive cells exposed to high H₂O₂ concentrations weredetached, floating and had disrupted cytoskeletons.

[1110] The maintenance of cell viability in the presence of lowconcentrations H₂O₂ is consistent with data from other laboratories. Forexample, Schorer et al (1985) showed that human umbilical veinendothelial cells (HUVEC) treated with 15 μM H₂O₂ averaged 90 to 95%viability (reported as 5% to 10% injury), whilst those exposed to 1500μM H₂O₂ were only 0%-50% viable (50% to 100% injured).

[1111] The use of H₂O₂ to mimic the tumor environment in vitro is alsoappropriate in that the tumor environment is rich in inflammatory cells,such as macrophages, PMNs and granulocytes, which produce H₂O₂ and otherreactive oxygen species. Although never before connected with stabletumor vascular markers, inflammatory cells are known to mediateendothelial cell injury by mechanisms involving reactive oxygen speciesthat require the presence of H₂O₂ (Weiss et al, 1981; Yamada et al.,1981; Schorer et al, 1985). In fact, studies have shown that stimulationof PMNs in vitro produces concentrations of H₂O₂ sufficient to causesublethal endothelial cell injury without causing cell death (measuredby chromium release assays) or cellular detachment; and that these H₂O₂concentrations are attainable locally in vivo (Schorer et al., 1985).

[1112] The present in vitro translocation data correlates with theearlier results showing that anti-PS antibodies localize specifically totumor vascular endothelial cells in vivo, and do not bind to cells innormal tissues. The finding that in vivo-like concentrations of H₂O₂induce PS translocation to the endothelial cell surface withoutdisrupting cell integrity has important implications in addition tovalidating the original in vivo data and the inventors' therapeuticapproaches.

[1113] Human, bovine and murine endothelial cells are all known to bePS-negative under normal conditions. Any previously documented PSexpression has always been associated with cell damage and/or ceildeath. This is not the case in the present studies, where normalviability is maintained. This shows that PS translocation in tumorvascular endothelium is mediated by biochemical mechanisms unconnectedto cell damage. This is believed to be the first demonstration of PSsurface expression in morphologically intact endothelial cells and thefirst indication that PS expression can be disconnected from theapoptosis pathway(s). Returning to the operability of the presentinvention, these observations again confirm that PS is a sustainable,rather than transient, marker of tumor blood vessels and a suitablecandidate for therapeutic intervention.

[1114] 2. Induction by Thrombin

[1115] Thrombin was also observed to increase PS expression, althoughnot to the same extent as H₂O₂. This data is also an integral part ofthe tumor-induction model of PS expression developed by the presentinventors: thrombin-induced PS surface expression in normal tissueswould also further coagulation as PS expression coordinates the assemblyof coagulation initiation complexes.

[1116] The tumor environment is known to be prothrombotic, such thattumor vasculature is predisposed to coagulation (U.S. Pat. No.5,877,289). As thrombin is a product of the coagulation cascade, it ispresent in tumor vasculature. In fact, the presence of thrombin inducesVCAM expression, contributing to the inventors' ability to exploit VCAMas a targetable marker of tumor vasculature (U.S. Pat. Nos. 5,855,866;5,877,289). The present data showing that thrombin also induces PSexpression is thus both relevant to targeting aminophospholipids withnaked antibodies and therapeutic conjugates, and further explains thebeneficial effects of the anti-VCAM coaguligand containing Tissue Factor(Example I).

[1117] 3. Other Agents of Oxidative Stress

[1118] Mouse bEnd.3 or bovine ABAE cells in vitro were treated for 24 hwith various concentrations of factors and conditions that are presentin the microenvironment of many tumors (Lichtenbeld et al., 1996; Harriset al., 1996), such as hypoxia/reoxygenation, thrombin, acidity,inflammatory cytokines and hydrogen peroxide (Table 11).

[1119] Externalization of PS and anionic phospholipids was quantified bymeasuring ¹²⁵I-annexin V binding. The amount of annexin V binding wascompared with that of cells in which apoptosis of 90-100% of cells hadbeen induced by combined treatment with actinomycin D and TNF-α.Actinomycin D and TNF-α induced the binding of 6.2 pmoles of annexin Vper 10⁶ cells (3.8×10⁶ molecules of annexin V per cell) on both celltypes, in good agreement with literature reports (Rao et al., 1992).This value was taken as the maximal level of externalized anionicphospholipids. TABLE 11 Induction of PS by Recreating Tumor Environment¹²⁵I-Annexin V (% of Max binding) Treatment Concentration ABAE CELLSbEnd.3 cells Medium with 10% serum N/A 0 0 Actinomycin D + TNFα 50 ng/mleach 100 100 VEGF 20 ng/ml 0 0 FGF-2 20 ng/ml 0 0 Scatter factor 40ng/ml 0 0 TGF β₁ 20 ng/ml 0 0 PDGF-BB 20 ng/ml 0 0 IL-10 20 ng/ml 0 0IL-8 20 ng/ml 0 0 IL-6 20 ng/ml 0 0 IL-1α 10 ng/ml 6.4 7.5 IL-1β 10ng/ml 5.8 5.5 Interferon 40 ng/ml 8.6 2.8 TNFα 20 ng/ml 7.4 13.7Thrombin 50 nM 8.8 17.4 Hypoxia 1% O₂ 15.0 to 17.5 22.5 Hypoxia + IL-1αSame as above 26.0 31.0 Hypoxia + TNFα Same as above 33.0 36.0 pH 6.6N/A 20.2 18.9 Hydrogen peroxide 200 μM 95.5 98.4

[1120] In Table 11, the concentrations of cytokines, growth factors andthrombin used were selected from literature values to have maximalstimulatory effect on cultured endothelial cells. These concentrationsdid not cause toxicity over the period of the test (24 h) as judged bymorphological appearance, a lack of detachment, and a lack of uptake oftrypan blue. The concentration of H₂O₂ employed was the maximalconcentration that did not cause cytotoxicity under the chosenconditions.

[1121] The basal binding of ¹²⁵I-annexin V was determined in thepresence of growth medium alone. Maximal PS exposure was determinedafter induction of apoptosis by the combined treatment with actinomycinD and TNF α. Average of duplicates from three separate studies ispresented. Standard error was less than 5%.

[1122] Untreated cells were largely devoid of externalized PS, as judgedby annexin V or anti-PS (9D2) antibody binding (Table 11). The basalbinding in the presence of growth medium alone was 0.44 and 0.68 pmolesof ¹²⁵I-annexin V for ABAE and bEnd.3 cells, respectively. Thiscorresponds to approximately 7.1% and 10.9% of the maximal binding forABAE and bEnd.3 cells, respectively, which correlated well with thefinding that approximately 10% of cells bound biotinylated annexin Vunder the same conditions.

[1123] VEGF, HGF, FGF, TGFβ₁, PDGF, IL-6, IL-8 and IL-10 did notincrease binding of ¹²⁵I-annexin V above the basal level for untreatedcells. Inflammatory mediators (IL-1α. IL-1β, TNFα and interferon) causeda small but reproducible increase in PS and anionic phospholipidtranslocation that ranged from 5 to 8% of the maximal level for ABAEcells and from 3 to 14% for bEnd3 cells.

[1124] Hypoxia/reoxygenation, thrombin or acidic external conditions (pH6.8-6.6) induced a moderately high externalization of PS and anionicphospholipid that ranged from 8 to 20% of the maximal level for ABAEcells and from 17 to 22% of the maximal level for bend.3 cells. Thelargest increase in PS and anionic phospholipid translocation wasobserved after treatment with 100 to 200 μM of hydrogen peroxide. Thistreatment caused nearly complete (95%) externalization of PS in bothcell types as judged by ¹²⁵I-annexin V binding (Table 11). More than 70%of ABAE and bEnd.3 cells bound biotinylated annexin V, as judgedimmunohistochemically.

[1125] Endothelial cells in which PS and anionic phospholipidtranslocation was generated by treatment with hypoxia/reoxygenation,thrombin, acidity, TNFα. IL-1 or H₂O₂ remained attached to the matrixduring time period of the assay (24 h), retained cell-cell contact andretained their ability to exclude trypan blue dye. Normal PS and anionicphospholipid orientation was restored 24 to 48 h later in the majorityof the cells after the inducing-factor was removed, or the cultureconditions were returned to normal. These results indicate that mildoxidative stress, created by direct application of H₂O₂ or indirectly byhypoxia/reoxygenation, acidity, thrombin, or inflammatory cytokines,triggers a transient translocation of PS and anionic phospholipids onviable endothelial cells.

[1126] 4. Combined Effects of Inflammatory Cytokines andHypoxia/Reoxygenation

[1127] Enhanced PS and anionic phospholipid exposure was observed whenABAE and bEnd.3 celis were subjected to hypoxia/re-oxygenation in thepresence of IL-1α or TNFα. In the absence of the cytokines,hypoxia/reoxygenation increased PS-exposure by ABAE cells to 15%-17.5%of the maximum level for cells treated with apoptotic concentrations ofactinomycin D and TNFα. In the presence of sub-toxic concentrations ofIL-1α or TNFα, hypoxia/reoxygenation increased anionicphospholipid-exposure to 26% and 33% respectively of the maximum (FIG.5; Table 11). Comparison with the effect of cytokines in the absence ofhypoxia/reoxygenation indicates that the combination of cytokines andhypoxia/reoxygenation had a greater than additive effects onPS-exposure. Similar effects were observed on bEnd.3 cells.

[1128] Therefore, in the tumor environment, the exposure of PS andanionic phospholipids induced by hypoxia/re-oxygenation may be amplifiedby inflammatory cytokines and possibly by such other stimuli as acidityand thrombin.

[1129] These in vitro studies shed light on the mechanism of PS exposureon tumor endothelial cells in vivo. They show that various factorsinduce PS exposure on endothelial cells without causing cytotoxicity,which mimics the situation in tumors in vivo. Hypoxia followed byreoxygenation, acidity, and thrombin most increased PS exposure onviable endothelial cells. Inflammatory cytokines (TNFα and IL-1α) alsocaused a weak but definite induction of PS exposure.

[1130] These conditions are likely to be the major inducing stimuli intumors in vivo because: i) PS positive endothelium is prevalent in andaround regions of necrosis where hypoxia, acidity, thrombosed bloodvessels, and infiltrating host leukocytes are commonly observed; ii) thefinding that hypoxia/reoxygenation amplifies the weak PS-exposingactivity of TNFα and IL-1 on endothelial cells in vitro correlates withthe situation in vivo in tumors where hypoxia and cytokine-secretingtumor and host cells co-exist; iii) hypoxia/reoxygenation and thrombinhave been reported to generate reactive oxygen species (ROS) inendothelial cells through activation of NADPH oxidase-like membraneenzyme (Zulueta et al., 1995). ROS produced by malignant cells mightcontribute to endothelial cell injury (Shaughnessy et al., 1989).Hydrogen peroxide was the most powerful inducer of PS exposure oncultured endothelial cells found in the present study, providingindirect support for the involvement of ROS.

[1131] Externalized PS provides a negative phospholipid surface uponwhich coagulation factors concentrate and assemble. This may contributeto the procoagulant status on the tumor endothelium that has long beenrecognized. PS also provides an attachment site for circulatingmacrophages (McEvoy et al., 1986), T lymphocytes (Qu et al., 1996) andpolymorphonuclear cells that assist in leukocyte infiltration intotumors. Adherence of activated macrophages, polymorphonuclear cells andplatelets to PS on tumor endothelium may lead to further secretion ofreactive oxygen species and further amplification of PS exposure.

EXAMPLE VIII Anti-Tumor Effects of Annexin Conjugates

[1132] The surprising finding that aminophospholipids and anionicphospholipids are stable markers of tumor vasculature means thatantibody-therapeutic agent constructs can be used in cancer treatment.In addition to using antibodies as targeting agents, annexins, and otherspecific binding proteins, can also be used to specifically delivertherapeutic agents to tumor vasculature. The following data shows theanti-tumor effects that result from the in vivo administration ofannexin-TF constructs.

[1133] A. Methods

[1134] An annexin V-tTF conjugate was prepared and administered to nu/numice with solid tumors. The tumors were formed from human HT29colorectal carcinoma cells that formed tumors of at least about 1.2 cm³.The annexin V-tTF coaguligand (10 μg) was administered intravenously andallowed to circulate for 24 hours. Saline-treated mice were separatelymaintained as control animals. After the one day treatment period, themice were sacrificed and exsanguinated and the tumors and major organswere harvested for analysis.

[1135] B. Results

[1136] The annexin V-tTF conjugate was found to induce specific tumorblood vessel coagulation in HT29 tumor bearing mice. Approximately 55%of the tumor blood vessels in the annexin V-tTF conjugate treatedanimals were thrombosed following a single injection. In contrast, therewas minimal evidence of thrombosis in the tumor vasculature of thecontrol animals.

EXAMPLE IX

[1137] Anti-Tumor Effects of 3SB Anti-PS Antibodies

[1138] The present example shows the anti-tumor effects of anti-PSantibodies using syngeneic and xenogeneic tumor models. The 3SB antibodyused in this study binds to PS (and PA), but is essentially devoid ofreactivity with PE. This anti-PS antibody caused tumor vascular injury,accompanied by thrombosis, and tumor necrosis.

[1139] The effects of anti-PS antibodies were first examined insyngeneic and xenogeneic tumor models using the 3SB antibody. For thesyngeneic model, 1×10⁷ cells of murine colorectal carcinoma Colo 26(obtained from Dr. Ian Hart, ICRF, London) were injected subcutaneouslyinto the right flank of BALB/c mice. In the xenogeneic model, a humanHodgkin's lymphoma L540 xenograft was established by injecting 1×10⁷cells subcutaneously into the right flank of male CB17 SCID mice. Tumorswere allowed to grow to a size of about 0.6-0.9 cm³ before treatment.

[1140] Tumor-bearing mice (4 animals per group) were injected i.p. with20 μg of 3SB anti-PS antibody (IgM), control mouse IgM or saline.Treatment was repeated 3 times with a 48 hour interval. Animals weremonitored daily for tumor measurements and body weight. Tumor volume wascalculated as described in Example I. Mice were sacrificed when tumorshad reached 2 cm³, or earlier if tumors showed signs of necrosis orulceration.

[1141] The growth of both syngeneic and xenogeneic tumors waseffectively inhibited by treatment with 3SB anti-PS antibodies (FIG. 6Aand FIG. 6B). Anti-PS antibodies caused tumor vascular injury,accompanied by thrombosis, and tumor necrosis. The presence of clots anddisintegration of tumor mass surrounding blocked blood vessels wasevident.

[1142] Quantitatively, the 3SB anti-PS antibody treatment inhibitedtumor growth by up to 60% of control tumor volume in mice bearing largeColo 26 (FIG. 6A) and L540 (FIG. 6B) tumors. No retardation of tumorgrowth was found in mice treated with saline or control IgM. No toxicitywas observed in mice treated with anti-PS antibodies, with normal organspreserving unaltered morphology, indistinguishable from untreated orsaline-treated mice.

[1143] Tumor regression started 24 hours after the first treatment andtumors continue to decline in size for the next 6 days. This wasobserved in both syngeneic and immunocompromised+tumor models,indicating that the effect was mediated by immune status-independentmechanism(s). Moreover, the decline in tumor burden was associated withthe increase of alertness and generally healthy appearance of theanimals, compared to control mice bearing tumors larger than 1500 mm³.Tumor re-growth occurred 7-8 days after the first treatment.

[1144] The results obtained with anti-PS treatment of L540 tumors arefurther compelling for the following reasons. Notably, the tumornecrosis observed in L540 tumor treatment occurred despite the fact thatthe percentage of vessels that stained positive for PS in L540 tumorswas less than in HT 29 and NCI-H358 tumors. This implies that even morerapid necrosis would likely result when treating other tumor types.Furthermore, L540 tumors are generally chosen as an experimental modelbecause they provide clean histological sections and they are, in fact,known to be resistant to necrosis.

EXAMPLE X

[1145] Anti-Tumor Effects of Antibody (9D2) Against AnionicPhospholipids

[1146] This example demonstrates the effects of the 9D2 antibody, whichbinds to PS and other anionic phospholipids, in anti-tumor studies invivo.

[1147] A high dose (>150 μg) of the rat antibody that binds to anionicphospholipids, 9D2, was injected into nude mice bearing H358 tumors.Immunolocalization studies shows that it strongly localized to tumorendothelium (4+), although some low level, non-specific binding of 9D2by normal vessels was observed due to the high dose (as would beobserved for a control IgM antibody of irrelevant specificity).

[1148] When 9D2 was injected i.p. into a SCID mouse with an L540 tumorfor ascites production, the tumor became necrotic and collapsed. Uponinjection of a control antibody (MK 2.7, rat IgG) into a SCID mouse withan L540 tumor, no similar effects were observed.

[1149] The effect of the 9D2 anti-PS antibody on the growth of L540tumors in vivo was then determined more precisely. Treatment was startedwhen tumors reached 200-250 μl (day 0). From day 0 to day 7, mice wereinjected i.p. with ˜150 μg of IgM (200 μl supernatant) or 200 μl of 10%DMEM. From day 7 to day 22, mice were injected i.p. with ˜300 μg of IgM(400 μl supernatant) or 400 μl of 10% DMEM. Day 22 was the last day oftreatment and the mice were sacrificed.

[1150] As shown in Table 12, from days 10 to 22, tumor growth isgenerally inhibited by about 40% to 50%. At the end of the study, only 4mice in the treated group have tumors larger than 2000 μl in volume, incontrast to 9/9 in the control group. TABLE 12 Effects of Anti-PSAntibodies on L540 Tumors In Vivo Day after Average Tumor Number of micewith start of the Volume (μl) % tumor volume >2000 μl treatment ControlTreated Inhibition Control Treated 0 341 320 6.2 0 0 1 464 325 10.8 0 03 412 415 0 0 0 7 687 455 33.8 0 0 10 904 544 39.9 1/9 0 13 945 545 42.41/9 0 15 1373 685 50.1 4/9 1/10 17 1426 842 41.0 4/9 4/10 20 1992 98750.5 6/9 4/10 22 2560 1365 53.3 9/9 4/10

[1151] In another in vivo study, the effects of the rat anti-PS antibodyon the growth of L540 tumors in CB17 SCID mice were followed for 45 daysafter tumor cell injections. These tumor-bearing mice were treated with300 μg of anti-PS antibody daily, i.p. or with 300 μl of 10% DMEM daily,i.p., as a control. Various parameters of tumor treatment were markedlybetter in the treated group in comparison to those of the controls(Table 13). TABLE 13 Effects of Anti-PS Antibodies on L540 Tumors InVivo Other parameters Control Treated % Regressed tumors¹ 0 40% (60 dayspost treatment) % Regressed tumors¹ 0 20% (90 days post treatment)Average volume of secondary 537 ± 30 366 ± 56 tumors (μl)²

[1152] In a further study, the 9D2 antibody was injectedintraperitoneally at a dose of 100 μg 3 times per week to mice with L540tumors. The tumor size was measured with calipers twice a week. Theanti-tumor effects in comparison to the control group is shown in FIG.7. The numbers in parenthesis indicate the number of mice with regressedtumors per total number of mice per group.

EXAMPLE XI

[1153] Anti-Tumor Effects of Anti-PS Antibody 3G4

[1154] The present example demonstrates additional anti-tumor effectsusing the anti-PS antibody 3G4 in syngeneic and xenogeneic tumor models.The 3G4 antibody used in this study is an IgG antibody that binds to PSand other anionic phospholipids (Example IV).

[1155] A. Protocols for Animal Tumor Studies

[1156] The effects of 3G4 was examined in syngeneic and xenogeneic tumormodels. The general protocol for the animal tumor treatment studies isconducted as follows. Unless particular differences are specified, thisis the protocol used throughout the studies of the present application.

[1157] The animals are obtained from Charles Rivers laboratories. Themice are 4-5 weeks, female, C.B-17 SCID or Fox Chase SCID mice. Mice arehoused in autoclaved caging, sterile food and water, with sterilehandling. All procedures performed in laminar flow hoods. Mice areacclimated 1 week and then ear-tagged and a blood sample (approximately75-100 μl) taken from the tail vein to check for leakiness by ELISA. Anymice that fail the leakiness ELISA test should not be used for testprocedures. Mice are injected orthotopically with tumor cells intomammary fat pad (MFP) or subcutaneously into the right flank 2-3 dayspost ear-tagging and blood sample removal.

[1158] In the orthotopic model, 1×10⁷ cells in 0.1 ml DMEM are typicallyinjected into MFP of anesthetized mice. Mice are anesthetized with 0.075ml of mouse cocktail injected IP. The mouse cocktail is 5 ml Ketamine(100 mg/ml); 2.5 ml Xylazine (20 mg/ml); 1 ml Acepromazine (10 mg/ml);11 ml sterile water. Dosage is 0.1 ml per 20-30 grams body weight viathe IP route for a duration of 30 minutes.

[1159] Once the mouse is anesthetized, as measured by no response totoe/foot pinch, the mouse is laid on its left side and wiped with 70%ethanol just behind the head and around the right forearm/back area. A2-3 mm incision is made just behind the right forearm (lateral thorax),which reveals a whitish fat pad when the skin flap is raised. 0.1 ml ofcells are injected into the fat pad using a 1 ml syringe and a 27-gaugeneedle, producing a bleb in the fat pad. The incision is closed using a9 mm sterile wound clip. The mouse is returned to its cage and observeduntil it has wakened from anesthesia and is mobile. Post-operativehealth status is determined, and if any signs of distress are observed,the animal is given acetaminophen (0.24 mg/ml)+codeine (0.024 mg/ml) inthe drinking water. The wound clip is removed after 1 week. This methodis used so that the cells are accurately placed into the selected siteand not into the subcutaneous region. Tumors will be approximately 200μl in volume (L×W×W) in 14-15 days and the take rate is essentially100%.

[1160] In the subcutaneous model, mice are typically injected with 1×10⁷cells in 0.2 ml. Mice are not anesthetized, but are restrained using asteady grip of mouse skin exposing the right flank. A 1 ml syringe witha 23 gauge needle is used to inject 1×10⁷ cells in 200 μl, just underthe skin of the mice and a bleb will be seen. It is not unusual toobserve a small amount of fluid leak from the injection site. A twistingmotion may be used when withdrawing the needle from the subcutaneousinjection to reduce this leakage. Tumor volume is measured by L×W×H.

[1161] In the perfusion protocol, mice are injected IV with 1000 U ofheparin in 0.2 ml saline. Mice are then be sedated by injecting themouse IP with 0.1 ml mouse cocktail. Once the mouse is sedated enough,as measured by no reflex when toe/foot is pinched, the thoracic cavityis opened to expose the heart and lungs. A 30 gauge needle attached totubing and perfusion pump is inserted into the left ventricle. The rightventricle is snipped so that blood can drip out. Saline is pumpedthrough for 12 minutes at a speed of 1 ml per minute. At the end of theperfusion, the needle and tubing are removed. Tissues are removed forfurther studies, either immunohistochemistry or pathology.

[1162] B. Tumor Treatment Results

[1163] For the syngeneic model, Meth A mouse fibrosarcoma tumor cellswere used. In one xenogeneic model, human MDA-MB-231 breast tumor cellswere seeded into the mammary fat pad. In another xenogeneic model, alarge human Hodgkin's lymphoma L540 xenograft was established byinjecting cells and allowing the tumor to grow to a size of over 500 mm³before treatment. Tumor-bearing mice (10 animals per group) wereinjected i.p. with 100 μg of 3G4 anti-PS antibody (IgG) as opposed tocontrol. Treatment was repeated 3 times a week. Animals were monitoredtwice a week for tumor measurements.

[1164] The growth of both syngeneic and xenogeneic tumors waseffectively inhibited by treatment with 3G4 anti-PS antibodies.Treatment for the first 20 to 30 days is shown in FIG. 8A, FIG. 8B andFIG. 8C. The antibodies caused tumor vascular injury, localizedthrombosis and tumor necrosis.

[1165] The treatment of the syngeneic, Meth A tumor cells wasparticularly successful, and the treatment of the human MDA-MB-231breast tumor cells growing in the mammary fat pad also produced tumorregressions (FIG. 8A and FIG. 8B). Even in mice bearing large L540tumors, known to be resistant to necrosis, the 3G4 antibody treatmentinhibited tumor growth in comparison to control. No retardation of tumorgrowth was found in control mice. No toxicity was observed in micetreated with anti-PS antibodies.

[1166] Tumors were also established using MD-MBA-435s cells and treatedas described above. The growth of these tumors was also effectivelyinhibited by treatment with the 3G4 antibody. The treatment of largeL540 tumors, MDA-MB-231 and MD-MBA-435s tumor cells for 60 days is shownin FIG. 8D, FIG. 8E and FIG. 8F. The antibodies caused tumor vascularinjury, thrombosis and necrosis and retarded tumor growth, with noevidence of toxicity.

[1167] MD-MBA-435s lucerifase cells were obtained from from Dr. AngelsSierra Jimenez, Barcelona, Spain and were grown in 10% DMEM. Mice wereinjected with tumor cells as described as above, and at 2 weeks postinjection, the tumors were measured and the volume recorded. Treatmentof mice with tumors of similar average volumes (200 mm³) was performedusing the 3G4 antibody and the chimeric 3G4 antibody, produced asdescribed in Example XIX, versus control. Treatment was initiated by IPinjection (800 μg) at day 15 and continued with injections of 200 μgevery two to three days until the final injection of 400 μg at day 35.Tumor volumes and mouse body weights were measured on injection days.Mice were sacrificed and perfused with saline for 12 minutes. The organsand tumor were removed, snap-frozen in liquid nitrogen and the tumorsectioned for immunohistochemistical analysis.

[1168] This study showed that both the 3G4 antibody and the chimeric 3G4antibody effectively retarded tumor growth as opposed to control (FIG.8G).

EXAMPLE XII

[1169] Anti-Viral Effects of Anti-PS Antibodies Against CMV

[1170] Surprisingly switching fields from tumor vasculature to viralinfections, the inventors next reasoned that antibodies toaminophospholipids and anionic phospholipids would also likely exert ananti-viral effect. The present example indeed shows this to be true,first using the 3G4 antibody in the treatment of cytomegalovirus (CMV)infection.

[1171] A. Methods

[1172] 1. Treatment of CMV-Infected Cells In Vitro

[1173] Confluent monolayers of human diploid foreskin fibroblasts(HHF-R2) in 6-well plates were infected with human CMV AD169 expressinggreen fluorescent protein (GFP) at an MOI=0.01 as previously described(Bresnahan et al., 1996). Briefly, the cells were incubated with virusin a total volume of 1 ml per well at 37° C. for 90 minutes. During theinfection, the plates were gently rocked every 30 minutes. Following theinfection, the cell supernatant was removed and DMEM/10% FBS/pen-strep(2 ml per well) was added to each well.

[1174] Dilutions of 3G4 or the isotype matched control antibody GV39G(100 μg/ml and 50 μg/ml) were added to the wells. The infected cellswere incubated at 37° C. for a total of 19 days. The medium and antibodyin each well was replaced every 3 days. On day 19, the cells andsupernatants from each well were harvested and frozen at −80° C. untilthe plaque assays were carried out.

[1175] 2. Fluorescent Microscopy

[1176] The recombinant CMV expresses GFP under the control of the SV40promoter. Hence, infected cells appear green under a fluorescentmicroscope. In these studies, the antibody treated CMV-infected cellswere observed under a fluorescent microscope at days 2, 3 and 9.

[1177] 3. Piaque Assays

[1178] The plaque assays were carried out using standard protocols.Briefly, the frozen cells cell suspensions were thawed quickly at 37° C.and centrifuged to remove debris at 1000 rpm for 1 minute. Differentdilutions of the cell supernatants were added to sub-confluentmonolayers of HHF-R2 cells in 6-well plates and the cells incubated at37° C. for 90 minutes (the plates were gently rocked every 30 minutes).Following the infection, the cell supernatants were removed and replacedwith 2 ml of DMEM/10% FBS. On day 4, the supernatant in each well wasremoved and the cells overlayed with 0.01% low melting pointagarose/DMEM/10% FBS. The plates were incubated at 37° C. for a total of14 days post-infection. On day 14, the infected monolayers were fixedwith 10% buffered formalin and stained with methylene blue to visualizethe plaques.

[1179] B. Results

[1180] 1. 3G4 Inhibits Viral Spread of CMV

[1181] To investigate whether 3G4 has an inhibitory effect on CMVinfection and replication, confluent human fibroblasts were pretreatedwith 3G4 before CMV was added at a low m.o.i. The CMV used in thesestudies expresses green fluorescent protein (GFP). Hence, infected cellsappear green when observed under a fluorescence microscope.

[1182] On day 3 of treatment, with both 50 μg/ml and 100 μg/ml ofantibody, there are single infected cells both in untreated wells and inwells treated with 3G4 or isotype-matched control antibody, GV39G. Thus,treating the fibroblasts with 3G4 does not appear to significantlyinhibit the entry of the virus into the cells.

[1183] On day 9, however, there is a dramatic difference in the numberof infected cells in 3G4-treated vs. control, GV39G-treated wells (FIG.9A and FIG. 9B; compare top right panel to middle and bottom rightpanels). While the virus has spread to approximately 80% of themonolayer in the control wells, the virus is restricted to the originalsingly-infected cell in the 3G4-treated wells. Hence, 3G4 limits thespread of CMV from the original infected cell to the surrounding cells.This inhibition of viral spread is observed when cells are treated with100 μg/ml (FIG. 9A) and 50 μg/ml (FIG. 9B).

[1184] 2. Viral Inhibition is Antibody Concentration-Dependent

[1185] In order to determine what concentration of 3G4 is necessary forthe anti-viral effect at a low m.o.i., infected cells were treated withdifferent concentrations of 3G4 and the control antibody, GV39G. Asshown in FIG. 10, the complete inhibition of cell-to-cell spread isobserved with 3G4 at 100 μg/ml and 50 μg/ml. When the cells were treatedwith 25, 12.5 and 6.25 μg/ml of 3G4, there are increasing numbers of GFPpositive CMV-infected cells. Although 3G4 does not totally prevent viralspread from the primary infected cells at these lower concentrations, itstill has a meaningful anti-viral effect, since fewer GFP-positiveCMV-infected cells are seen in the 3G4-treated well as compared toGV39G-treated, control wells (FIG. 10).

[1186] 3. Quantification of Viral Load at a Low M.O.I.

[1187] The anti-viral effect of 3G4 was quantitated by carrying outplaque assays to determine the viral load following antibody-treatment.The controls included untreated cells, the GV39G antibody and anadditional antibody control using the C44 antibody, a mouse IgG2aisotype antibody to colchicine.

[1188] Treatment of infected cells (m.o.i.==0.01 pfu/cell) with 100μg/ml of 3G4 resulted in a dramatic 6 log₁₀ decrease in viral titer ascompared to control, GV39G-treated cells (FIG. 1I A). This inhibitiontranslates into an approximately 99.9999% inhibition of viralreplication. At a concentration of 50 μg/ml, treatment with 3G4 resultsin a 3.5 log₁₀ decrease in viral titer as compared to GV39G-treatment.Using 3G4 at 25 μg/ml and 12.5 μg/ml, the results are still dramatic,and even at 6.25 μg/ml an inhibitory effect is still observed (FIG.11A).

[1189] 4. Quantification of Viral Load at a High M.O.I.

[1190] 3G4 treatment of fibroblasts infected at a high m.o.i. of 3 alsoresults in a dramatic reduction in viral titer. At 100 μg/ml, treatmentwith 3G4 resulted in a 5 log₁₀ decrease in viral titer as compared tocontrol, GV39G-treated cells (FIG. 11B). At 50 μg/ml, 3G4 inhibitedviral replication by 3 logs when compared to GV39G (FIG. 1I B).

[1191] 5. Inhibition of Replication at a Late Stage

[1192] To determine which stage of the CMV replicative cycle is blockedby 3G4, a timed addition study was performed. For this, 3G4 was added tofibroblasts infected at a high m.o.i. at different time points after theinfection. The viral load (in both the cells and supernatant) wasquantified using a standard plaque assay.

[1193] Addition of 3G4 up to 24 hours after infection resulted in a 5-6log₁₀ decrease in viral titer (FIG. 1I C). However, when addition of 3G4was delayed to 48 hours, the inhibitory effect of 3G4 was reduced to 2log₁₀ and when addition was delayed to 72 to 96 hours, the inhibitoryeffect was further reduced. This shows that 3G4 interferes with a latestage of CMV replication that occurs between 24-48 hours afterinfection. Thus, 3G4 does not significantly interfere with infection orwith immediate early or early gene expression. It rather acts later inthe viral replication cycle, e.g., on late gene expression, viral DNAsynthesis, viral packaging or egress.

EXAMPLE XIII Anti-Viral Effects of Anti-PS Antibodies Against RSV

[1194] In addition to the dramatic anti-viral effects against CMV shownin Example XII, the present example demonstrates the use of threedifferent anti-PS antibodies in the inhibition of Respiratory SyncitialVirus (RSV) replication.

[1195] A. Methods

[1196] 1. Treatment of RSV-Infected Cells In Vitro

[1197] A-549 cells were grown to 100% confluence in three Costar 12-welltissue culture plates. 200 μL of minimum essential Eagle medium wasadded to all wells. Anti-phospholipid antibody (Ab) was added (100 μg in100 μL) to 9 wells of each plate and 30 min. later cells in 6 of thoseinitial 9 wells were infected with an MOI of 1 with RSV long strain in avolume of 100 uL. The three remaining wells were left as non-infected,antibody-treated wells. The three other wells with no antibody wereinfected with RSV at the same MOI as described above.

[1198] Each plate was used to test the three different antibodies: 3G4,3SB and 1B9 (Example IV). Cells were incubated in 5% CO₂ at 40° C. for 2hours and then 600 μL of medium was added to complete 1 mL volume ineach well. An A-549 cell plate was kept in the same conditions, ascontrol. Supernatants were collected at 4, 24 and 72 hours afterinfection. At each time point, four wells from each plate were sampled:one well with only-Ab treated cells, two wells hadAb-treated/RSV-infected cells and one well had RSV-infected only cells.The samples were frozen at −80 until the plaque assay.

[1199] 2. Plaque Assays

[1200] The plaque assays were carried out as previously described (Kischet al., 1963; Graham et al., 1988). Briefly, the frozen cells cellsuspensions were thawed quickly at 37° C. Three 10-fold dilutions weremade from the undiluted cell supernatants: 10⁻¹, 10⁻², and 10⁻³. 100 μLof each dilution plus the undiluted sample were inoculated into 80%confluent Hep-2 cell line plates, all in triplicates. Plates were placedin the 5% CO₂, 40° C. incubator for 5 days. On the 5^(th) day, theplates were developed and stained with hematoxylin and eosin to revealthe plaques in each well. The plaques were counted using a dissectingmicroscope to calculate the RSV viral load in pfu (plaque formingunits)/mL.

[1201] B. Results

[1202] As seen in FIG. 12, treatment of RSV-infected cells with either3SB or 1B9 resulted in a log decrease in viral replication. Theanti-viral effect was even more pronounced when the infected cells weretreated with 3G4. Treatment with 3G4 resulted in a 2 log₁₀ decrease inviral titer (FIG. 12). The inhibition was lower than seen with CMV, mostlikely because the concentration of 3G4 was low (25-50 μg/ml).

EXAMPLE XIV Single Chain Anti-PS Antibodies

[1203] Given the many uses of anti-PS antibodies described herein,including as anti-tumor agents alone, as targeting agents for deliveringattached therapeutic agents to tumors, and as anti-viral agents, thepresent example describes techniques suitable for generating singlechain (scFv) anti-PS antibodies, i.e., wherein the V_(H) and V_(L)domains are present in a single polypeptide chain, generally joined by apeptide linker.

[1204] A. Preparation of the Phage Antibody Library

[1205] The secondary stock of the bacterial library (about 1×10¹⁰clones) was inoculated into 100 ml 2×TY containing 100 μg/ml ampicillinand 1% glucose. It was grown with shaking at 37° C. until the OD at 600nm was 0.5.

[1206] M13KO7 helper phage was added at 10¹³ pfu and incubated withoutshaking in a 37° C. water bath for 30 min. The infected cells werecentrifuged at 3,500 g for 10 min. The pellet was resuspended in 200 mlof 2×TY containing 100 μg/ml ampicillin and 75 μg/ml kanamycin andincubated with shaking at 30° C. overnight.

[1207] The culture was centrifuged at 10,800 g for 10 min. 1/5 volumePEG/NaCl was added to the supernatant, mixed well and left for 1 hr at4° C. It was then centrifuged at 10,800 g for 30 min. The pellet wasresuspended in 40 ml PBS and 8 ml PEG/NaCl was added. It was mixed andleft for 20 min at 4° C. It was then centrifuged at 10,800 g for 10 minand the supernatant aspirated. The pellet was resuspended in 2 ml 10%human serum and centrifuged at 11,600 g for 10 min in a microcentrifugeto remove most of the remaining bacterial debris.

[1208] To pre-pan, the phage antibody library in 10% human serum wasadded to the PC coated dish and incubated for 60 min at roomtemperature.

[1209] B. Selection on Biotinylated Liposomes

[1210] 20 μmol phosphatidylinositol and 20 μmol biotinylatedphosphatidylserine were dissolved in 10 ml hexane. This solution wasdried to a thin layer on the surface of a flask using a rotatingevaporator. 2 ml PBS was added and bath sonicated 4° C. for 30 minutes.

[1211] 100 μl phage scFv and 100 μl biotinylated liposomes were thenmixed in the presence of 10% human serum and gently rotated for one hourat room temperature. Blocking was done with 100 μl streptavidin M-280dynabeads by adding 600 μL 2.5% casein/0.5% BSA for 30 min at roomtemperature. The beads were separated from the blocking buffer with aMPC-E (Magnetic Particle Concentrator from Dynal) for 4-5 min.

[1212] The beads were resuspended in 100 μl PBS. 100 μl of blockedstreptavidin Dynabeads was added to the phage bound to the biotinylatedantigen and gently rotated for 15 min at room temperature. Separationwas achieved with a MPC-E for 5 minutes and the supernatant poured off.It was washed five times with 1 ml PBS. For each wash, the beads wereresuspended and brought down with a MPC-E.

[1213] Finally, the phage was eluted from the beads by resuspending in300 μl 100 mM triethalamine for 30 mins. 150 μl 1 M Tris pH=7.4 wasadded for neutralization. The beads were separated again with the MPC-E.

[1214] 150 μl of the phage supernatant was used to infect 10 ml TG1bacteria in log phase. The 10 ml culture was shaken in the presence of20 μg/ml ampicillin at 37° C. for one hour. Ampicillin was added to thefinal concentration of 50 μg/ml and shaken for another hour. 10¹³ pfuM13 helper phage was added to this culture, transferred to 100 ml 2TYmedium containing 100 μg/ml ampicillin and shaken at 37° C. for onehour. Kanamycin was added to the final concentration of 100 μg/ml andshaken at 30° C. overnight.

[1215] The phage preparation procedure was repeated and the selectionprocedure repeated another 3 to 4 times.

[1216] C. Monoclonal Single Chain Antibody ELISA

[1217] Individual HB2151 colonies from the plates (after 4 rounds ofselection) were inoculated into 500 μl 2×TY containing 100 μg/mlampicillin and 1% glucose in 96-well plates and grown with shaking (300rpm.) overnight at 37° C. 5 μl from this plate were transferred to asecond 96-well plate containing 500 μl 2×TY containing 100 μg/mlampicillin per well and grown shaking at 37° C. for 3 hr (OD600=0.9).

[1218] To each well was added 50 μl 2×TY containing 100 μg/mlampicillin, 10 mM IPTG (final concentration is 1 mM), which was grownwith shaking overnight at 30° C. It was centrifuged at 1,800 g for 10min and 100 μl of the supernatant used in the following ELISA.

[1219] 96 well plates (DYNEX IMMULON® 1B) were coated with PS dissolvedin ethanol at a concentration of 10 μg/ml (P6641 10 mg/ml solvent wasChloroform:MeOH 95:5). 10 μg/ml PC was coated in the same way. Theseplates were evaporated at 4° C. in the cold room. 250 μl 2.5% casein wasadded to each well, and the plates were covered and blocked at 37° C.for 1 hour.

[1220] Wells were rinsed 3 times with PBS, 100 μl/well 10% human serumand 100 μl/well supernatant containing soluble scFv was added andincubated for 60 min at 37° C. The solution was discarded and washed 6times with PBS. 100 μl 9E10 in 5% casein/0.5% BSA-PBS (1:5000 dilution)was added to each well, incubated at 37° C. for 1 hour and washed 6times with PBS. 100 μl HRP-goat-anti-mouse antibody (1:10000 dilution)was added to each well, incubated at 37° C. for 1 hour and washed 5times with PBS. 100 μl 0.05% OPD was added to each well and developedfor 5 minutes. 100 μl 0.18 M H₂SO₄ was added to stop the reaction andread at O.D. 490.

[1221] Antigen-positive clones were streaked on 2×TYAG plates and grownovernight at 30° C. Positive single colonies were picked into 3 ml2×TYAG media and grown 12 hours at 37° C. Plasmids were extracted andscFv gene inserts checked by enzyme digestion and PCR. The ones with thecorrect size inserts were sequenced.

[1222] The colonies with the correct size inserts were grown into 100 ml2×TYAG media and shaken at 37° C. OD 600=0.5. These were transferredinto 900 ml 2×TYA and grown until OD 600=0.9. 1 M IPTG was added to afinal concentration of 1 mM and shaken at 30° C. overnight. Thesupernatant was checked using the same ELISA method as previously. ThescFv protein was purified from the periplasmic fraction usingNi⁺⁺-agarose affinity chromatography.

[1223] D. Results

[1224] After 4 rounds of panning, the following clones gave promisingELISA signal on PS plates and have the correct size insert: 3E5, 3A2,G5, C8, E4 and 4D5. These have been subcloned, wherein E4 gave 5positive subclones and 4D5 gave 5 positive subclones (Table 14). TABLE14 ELISA on PS Plate 0.099 0.107 0.118 0.115 0.100 0.094 0.084 0.0860.166 0.164 0.102 0.191 0.113 0.106 0.127 0.150 0.128 0.097 0.078 0.0870.190 0.144 0.102 0.154 0.122 0.115 0.117 0.112 0.105 0.097 0.085 0.0880.230 0.071 0.168 0.150 0.107 0.108 0.121 0.123 0.107 0.101 0.083 0.0850.191 0.246 0.186 0.150 0.138 0.121 0.114 0.131 0.100 0.096 0.082 0.0790.183 0.187 0.275 0.171 0.118 0.115 0.116 0.132 0.099 0.094 0.082 0.0860.185 0.073 0.208 0.102 0.111 0.176 0.126 0.118 0.096 0.087 0.123 0.0870.144 0.226 0.112 0.126 0.102 0.107 0.131 0.125 0.089 0.102 0.082 0.0840.188 0.073 0.142 0.151 3E5 3A2 G5 C8 E4 4D5

[1225] Once the positive clones were identified, they were sequenced.The ScFv nucleic acid and protein sequence of clone 3A2 is set forth inSEQ ID NO:5 and SEQ ID NO:6, respectively. The positive clones weregrown up on a large scale and the scFv purified using Nickel agaroseaffinity chromatography. The purified scFv has been obtained usingPhast-gel electrophoresis.

EXAMPLE XV Synthesis of PE-Binding Peptide Derivatives

[1226] The present example concerns the design and synthesis ofexemplary PE-binding peptide derivatives and conjugates for use intreating tumors and viral diseases. The structures for exemplaryduramycin derivatives are set forth in the panels of FIG. 13A throughFIG. 13O, which match the following description.

[1227] A. DLB

[1228] 0.5 mg (0.25 μmole) of duramycin dissolved in 0.387 ml 0.1MNaHCO₃ in water was added to 0.113 mg (0.25 μmole) of NHS-LC-Biotin(Sigma). The reaction mixture was incubated at room temperature for 1 hrand then at 4° C. overnight. The sample was loaded onto a silica column,washed with 0.1% trifluoroacetic acid (TFA), eluted with 0.1% TFA and70% CH₃CN. The eluant was collected and concentrated by centrifugation.The total yield was 0.5 mg (FIG. 13A).

[1229] B. DIB

[1230] 0.5 mg (0.25 μmole) of duramycin dissolved in 0.286 ml of 0.1MNaHCO₃ in water was added to 0.034 mg (0.25 μmole) of 2-iminothiolanehydrochloride (2-IT). The mixture was incubated at room temperature for1 hr. 0.13 mg (0.26 μmole) of iodoacetyl-LC-Biotin (Pierce) was addedand the reaction incubated at room temperature for 1 hr and at 4° C.overnight. The sample was loaded onto a silica column, washed with 0.1%TFA, eluted with 0.1% TFA and 70% CH₃CN. The eluant was collected andconcentrated by centrifugation. The total yield was 0.5 mg (FIG. 13B).

[1231] C. (DLB)₄NA

[1232] 1.9 mg (0.94 μmole) duramycin was dissolved in 0.5 ml of 0.1MNaHCO₃ in water. To this, 0.4 mg (0.88 μmole) NHS-LC-Biotin (Sigma) in200 μl dimethylformamide (DMF) was added. The mixture was incubated atroom temperature for 4 hr. 10 mg (0.17 μmole) neutravidin (NA) in 1 mlwas added to the reaction mixture, which was incubated at roomtemperature for 2 hr and then at 4° C. overnight. The reaction mixturewas then loaded onto a G-25 column (volume 50 ml) in PBS buffer. Thefractions were collected and analyzed by SDS PAGE (phast gel).Protein-containing fractions (7-16) were pooled together, sterilized byfiltration through a 0.22 μm filter and the concentration determined bymeasuring absorption at 280 nm. The total yield was 5.1 mg.

[1233] The sample was then fractionated by FPLC. Three peaks werecollected that corresponded to the following: peak 1: [(DLB)₄NA]₃(fractions 17-23); peak 2: [(DLB)₄]₂ (fractions 24 33) and peak 3:(DLB)₄NA (fractions 35-48). All the samples were sterilized byfiltration through a 0.22 μm filter. The final yields obtained were:0.34 mg of [(DLB)₄NA]₃; 0.59 mg of [(DLB)₄]₂ and 1.41 mg of (DLB)₄NA(FIG. 13C).

[1234] D. (DLB)₄NA-F

[1235] 0.61 mg of (DLB)₄NA in PBS buffer was added to 0.005 mgN-hydroxysuccinimidyl fluorescein (NHS-Fluorescein) (Sigma) in DMF. Themixture was incubated at room temperature for 1 hr. The reaction mixturewas then fractionated on a PD10 column (10 ml). (DLB)₄NA-F was eluted inthe protein-containing fractions (3 and 4), which were pooled togetherand sterilized by filtration through a 0.22 μm filter. The total yieldwas 0.5 mg (FIG. 13D).

[1236] E. (DIM)_(n) HIgG

[1237] Human IgG (HIgG) was first purified as follows: 1.3 ml HIgG (thatincluded 100 mg/ml HIgG, 22.5 mg/ml glycine and 3 mg/ml albumin inborate buffer with 1 mM EDTA, pH 9) was applied to an FPLC (S200, 250ml) column. The fractions were collected and analyzed by SDS PAGE on aphast gel. Fractions containing monomeric IgG (21-32) were pooledtogether and sterilized by filtration through a 0.22 μm filter. Thetotal yield as determined by absorption at 280 nm was 111 mg.

[1238] Purified HIgG (55 mg in 13 ml of borate buffer, pH 9) was addedto 1.003 mg in 0.5 ml of SMCC (Pierce) in DMF. The mixture was incubatedat room temperature for 1 hr. At the same time, another reaction mixturecontaining 6 mg duramycin (3 μmole; dissolved in 0.5 ml 0.1M NaHCO₃) and0.413 mg 2-IT (3 μmole; in 0.1M NaCO₃) was incubated at room temperaturefor 1 hr. After completion of the reactions, the two reaction mixtureswere combined and incubated at room temperature for 2 hr and at 4° C.overnight. The reaction products were analyzed by SDS PAGE on a phastgel. The reaction products were loaded onto an FPLC column in boratebuffer, pH 9. The FPLC fractions corresponding to trimer (5-14), dimer(15-24), and monomer (25-37) were pooled and sterilized by filtrationthrough a 0.22 μm filter. The total yield of monomer was 54.6 mg. Fiveto seven duramycin groups were attached to each molecule of HigG (FIG.13E).

[1239] F. (DIM)_(n) HIgG-F

[1240] 1 mg (0.7 ml) of (DIM)_(n)HIgG was added to 5 μl ofNHS-Fluorescein in DMF. The reaction mixture was incubated at roomtemperature for 1 hr and desalted on a PD-10 column. Protein-containingfractions (2-3) were pooled and sterilized by filtration through a 0.22μm filter. The total yield was 0.9 mg (FIG. 13F).

[1241] G. (DIM)_(n) HIgG-B and [(DIM)_(n)HIgG]₂-B

[1242] To synthesize biotinylated derivatives of [(DIM)_(n)HIgG]₂, 0.66mg (1 ml) of [(DIM)_(n)HIgG]₂ was added to 811 of 1 mg/ml ofNHS-LC-Biotin (Pierce) in DMF. The mixture was incubated at roomtemperature for 1 hr. The reaction mixture was then desalted on a PD-10column. Protein-containing fractions (3 and 4) were pooled andsterilized by filtration through a 0.22 μm filter. The final yield was0.46 mg.

[1243] The biotinylation of the monomer (DIM)_(n)HIgG was performed inthe same manner. Briefly, 1.06 mg (0.75 ml) of (DIM)_(n)HIgG were addedto 12 μl of 1 mg/ml NHS-LC-Biotin in DMF. After incubation at roomtemperature for 1 hr, the reaction product was desalted on a PD-10column. Protein-containing fractions (3 and 4) were pooled andsterilized by filtration through a 0.22 μm filter. The final yield was0.62 mg (FIG. 13G).

[1244] H. (DIB)₄NA

[1245] 2 mg (0.99 μmole) of duramycin were dissolved in 0.5 ml 0.1MNaHCO₃ and added to 0.136 mg (0.99 μmole) of 2-IT. The reaction mixturewas incubated at room temperature for 1 hr. Following this, 0.483 mg(0.95 μmole) of iodoacetyl-LC-Biotin (Pierce) was added and the reactionmixture incubated at room temperature for 1 hr. 10 mg (0.17 μmole) ofneutravidin in 1 ml of H₂O was added and incubated at 4° C. overnight.The reaction mixture was fractionated by FPLC. Three different peakswere collected and pooled: [(DIB)₄NA]₃ (fractions 17-23); [(DIB)₄NA]₂(fractions 24-33); and (DIB)₄NA (fractions 35-48). All the samples weresterilized by filtration through a 0.22 μm filter. The total yieldsobtained were 0.87 mg of [(DIB)₄NA]₃; 1.25 mg of [(DIB)₄NA]₂; and 1.83mg of (DIB)₄NA (FIG. 13H).

[1246] I. (DIB)₄NA-B

[1247] 0.023 mg (0.3 μmole) of (DIB)₄NA was added to 0.9 μg ofNHS-LC-Biotin (Pierce). The reaction was incubated at room temperaturefor 1 hr and then desalted on a PD-10 column. The total yield was 0.04mg (FIG. 13I).

[1248] J. DS-1

[1249] 5 mg (2.5 μmole) of duramycin dissolved in 0.5 ml of 0.1M NaHCO₃in water was added to 0.319 mg (2.6 μmole) of 1,3 propane sultone. Themixture was incubated at 4° C. overnight. The sample was loaded onto asilica column, washed with 0.1% TFA, eluted with 0.1% TFA and 70% CH₃CN.The eluant was collected and concentrated by centrifugation underreduced pressure. The total yield was 5 mg (FIG. 13J).

[1250] K. DS-2

[1251] 1 mg (0.497 μmole) of duramycin dissolved in 0.3 ml of 0.1MNaHCO₃ in water was added to 0.072 mg (0.523 μmole) of 2-IT. Thereaction mixture was incubated at room temperature for 1 hr. 0.125 mg(0.49 μmole) of SBF-Chloride (Pierce) was added. The reaction mixturewas incubated at room temperature for 1 hr and 4° C. overnight. Thepeptide was purified on a silica column. The eluant was collected andconcentrated by centrifugation under reduced pressure. The total yieldwas 1 mg (FIG. 13K).

[1252] L. DS-3

[1253] 1 mg (0.497 μmole) of duramycin dissolved in 0.4 ml of 0.1MNaHCO₃ in water was added to 0.109 mg (0.592 μmole) of 2-sulfobenzoicacid cyclic anhydride. The reaction was incubated at room temperaturefor 1 hr and 4° C. overnight. The peptide was purified on a silicacolumn. The eluant was collected and concentrated by centrifugationunder reduced pressure. The total yield was 1 mg (FIG. 13L).

[1254] M. DS-4

[1255] 0.25 mg (0.124 μmole) of duramycin dissolved in 0.5 ml of 0.1MNaHCO₃ in water was added to 0.017 mg (0.124 μmole) of 2-IT. Thereaction mixture was incubated at room temperature for 1 hr. The mixturewas then added to 0.049 mg (0.124 μmole) Ellman's reagent. The mixturewas incubated at room temperature for 2 hr and overnight at 4° C. 250 μlof 1 mg/ml of 4-Amino-5-hydroxy-2,7-naphthalene disulfonic acidmono-sodium salt hydrate was added to 100 μl of 1 mg/ml 2-IT. Thereaction was incubated at room temperature for 1 hr. 50 μl of thisreaction mixture was added to the previous reaction and incubated atroom temperature for 1 hr. The peptide was purified on a silica column.The eluant was collected and concentrated by centrifugation underreduced pressure (FIG. 13M).

[1256] N. DS-5

[1257] 5 mg (2.5 mole) of duramycin dissolved in 0.5 ml of 0.1M NaHCO₃in water was added to 0.356 mg (2.6 μmole) of 1,3 butane sultone. Themixture was incubated at 4° C. overnight. The sample was loaded onto asilica column, washed with 0.1% TFA, eluted with 3.1% TFA and 70% CH₃CN.The eluant was collected and concentrated by centrifugation underreduced pressure. The total yield was 5 mg (FIG. 13N).

[1258] O. DC-1

[1259] 0.25 mg (0.124 μmole) of duramycin dissolved in 0.5 ml of 0.1MNaHCO₃ in water was added to 0.017 mg (0.124[mole) of 2-IT. The reactionmixture was incubated at room temperature for 1 hr. The mixture was thenadded to 0.049 mg (0.124 μmole) Ellman's reagent. The mixture wasincubated at room temperature for 2 hr and overnight at 4° C. Thepeptide was purified on a silica column. The eluant was collected andconcentrated by centrifugation under reduced pressure (FIG. 13O).

EXAMPLE XVI Duramycin Derivatives Specifically Bind PE

[1260] The present example shows that the duramycin derivativessynthesized in Example XV are specific for PE and can therefore be usedas designed, by linking to cell-impermeant, targeting or anti-viralagents and use in the treatment of tumors and viral diseases.

[1261] To test the specificity of the duramycin derivatives,particularly the binding to PE in preference to other phospholipids, aseries of competition ELISAs were performed. The ability of theduramycin derivatives to compete with either DIB or DLB for binding toPE was tested in the following method.

[1262] PE and PC were dissolved separately in ethanol. The finalconcentration was 5 μg/ml. 100 μl was added to each well of 96 wellELISA plates (DYNEX IMMULON® 1B). These plates were evaporated at 4° C.in a cold room. 250 μl 2.5% casein was added to each well, covered andblocked at 37° C. for 1 hour. The blocking buffer was discarded and 100μl 2.5% casein added to each well. The duramycin compound was added as aserial dilution across the plate, such as (DIM)nHIgG, (DIB)4NA,(DLB)4NA, DS, duramycin and DIB.

[1263] The (DIM)nHIgG starting concentration was 1.4 mg/ml, the (DIB)4NAstarting concentration was 800 μg/ml, and the (DLB)₄NA startingconcentration was 800 μg/ml. These were incubated at 37° C. for 1 hourand washed 5 times with PBS. 100 μl HRP-streptavidin (1:5000 dilution)was added to each well, incubated at 37° C. for 1 hour and washed 5times with PBS. 100 μl 0.05% OPD was added to each well and developedfor 5 minutes. 100 μl 0.18 M H2SO4 was added to stop the reaction andread at O.D. 490.

[1264] The resultant data was tabulated and then plotted graphically. Asexemplified by the data in FIG. 14C and FIG. 14D, increasingconcentrations of the duramycin derivatives decrease absorbance at 490nm, showing that the duramycin derivatives compete with DIB and DLB forbinding to phosphatidylethanolamine.

[1265] The phospholipid binding profiles of duramycin constructs wereconfirmed using further ELISAs. The respective test lipids PS, PE, PI,CL, PC, PG, SM, and cholesterol were dissolved separately in ethanol andused to coat ELISA plates. Duramycin compounds were added as serialdilutions across the plates. After incubation and washing steps, asecondary detection reagent was added to each well and reactivitydetermined using the colorimetric assay as described above.

[1266] Representative phospholipid binding profiles for the duramycinbiotin derivatives, DIB and DLB are depicted in FIG. 14A. It is shownthat DIB and DLB are specific for PE, with binding to each of PS, PI,CL, PC, PG and SM being negligible or undetectable, (DIM)_(n)HIgG-B and[(DIM)_(n)HIgG]₂-B had essentially the same binding profile as DLB.Although minimal binding to PS was observed at high concentrations ofDIB (FIG. 14A), this is not meaningful in the context of this study, asbinding to PS was undetectable at DIB concentrations that weresaturating and half maximal for PE binding. Therefore, the duramycinconstructs specifically bind to phosphatidylethanolamine.

[1267] It was also shown that serum has no significant effect on PEbinding by duramycin derivatives. This is exemplified by binding of theduramycin biotin derivative, DLB to PE-coated ELISA plates in thepresence and absence of serum (BSA), wherein the binding profiles showno significant differences (FIG. 14B).

EXAMPLE XVII Anti-Viral Effects of PE-Binding Peptide Derivatives

[1268] In addition to the anti-viral effects mediated by anti-PSantibodies, as shown in Example XII and Example XIII, the presentexample demonstrates the anti-viral effects of peptide derivatives thatspecifically bind to the other common aminophospholipid, PE.

[1269] A. Methods

[1270] 1. Treatment of CMV-Infected Cells In Vitro

[1271] Confluent monolayers of human diploid foreskin fibroblasts(HHF-R2) in 6-well plates were infected with human CMV AD169 expressinggreen fluorescent protein (GFP) at an MOI=0.01 as described in ExampleXII (Bresnahan et al., 1996). The cells were incubated with virus in atotal volume of 1.5 ml per well at 37° C. for 90 minutes. During theinfection, the plates were gently rocked every 30 minutes. Following theinfection, the cell supernatant was removed and DMEM/10% FBS/pen-strep(2 ml per well) was added to each well.

[1272] Different dilutions of duramycin derivatives (DLB)₄NA,(DIM)_(n)HIgG, DS-1, DS-2, DS-3 and DC-1 were added to the wells beforethe addition of the virus, and following infection. The infected cellswere incubated at 37° C. for a total of 14 days. The medium andduramycin derivative in each well were replaced every 3 days.

[1273] 2. Fluorescent Microscopy

[1274] As in Example XII, the recombinant CMV expresses GFP under thecontrol of the SV40 promoter. Hence, infected cells appear green under afluorescent microscope. In these studies, the CMV-infected cells treatedwith the duramycin derivatives were observed under a fluorescentmicroscope at days 4 and 6.

[1275] B. Results

[1276] On day 4, there are single infected GFP-positive green cells inuntreated wells and wells treated with (DLB)₄NA and (DIM)_(n)HIgG (FIG.15, left panels). Thus, treatment of HHF-R2 cells with these duramycinderivatives does not appear to inhibit the entry of the virus into thecells. There is some preliminary evidence that the duramycin derivativesDS-1, DS-2 and DS-3 inhibit viral entry into the cells.

[1277] On day 6 after treatment with (DLB)₄NA and (DIM)_(n)HIgG, thereis a marked difference in the number of infected GFP-positive cells inuntreated vs. the duramycin derivative treated wells (FIG. 15, middlepanels). By day 6, the virus has spread from the single infected cellseen on day 4 surrounding cells in the untreated wells (FIG. 15, top,compare left panel to middle panel). However, on day 6 in the wellstreated with (DLB)₄NA and (DIM)_(n)HIgG, the virus is limited to theoriginal singly infected cell (FIG. 15, middle and bottom, compare leftpanels to middle panels).

[1278] Accordingly, (DLB)₄NA and (DIM)_(n)HIgG limit the spread of CMVfrom the original infected cell to the surrounding cells. Thisinhibition of viral spread is observed when cells were treated withdifferent concentrations of (DLB)₄NA (100 μg/ml and 50 μg/ml) and(DIM)_(n)HIgG (200 μg/ml and 100 μg/ml).

EXAMPLE XVIII Advantages of 3G4 Antibody

[1279] The 3G4 antibody developed by the inventors' unique protocol, asdescribed in Example IV, has many advantages over the anti-PS antibodiesin the literature, including the prominent anti-PS antibody, 3SB (Roteet al. (1993). The present example describes certain of thoseadvantages.

[1280] A. Class and Specificity

[1281] 3G4 is an IgG antibody, whereas 3SB is IgM. Antibodies of IgGclass have numerous advantages over IgM, including higher affinity,lower clearance rate in vivo and simplicity of purification,modification and handling. A comparison of the PS binding of the IgMantibody, 3SB, with 3G4 and another IgG antibody is shown in FIG. 19Aand FIG. 19B.

[1282] 3G4 reacts strongly with the anionic phospholipids PS, PA, PI, PGand CL with approximately the same intensity, and binds to theaminophospholipid, PE less strongly. It has no reactivity with PC and SMand has the binding specificity profile: PS=PA=PI=PG>CL>>PE (Example IV;Table 4). 3G4 does not bind detectably to heparin, heparan sulfate or todouble or single stranded DNA, nor to cellular proteins extracted frombEnd.3 cells on Western blots. The binding of 3G4 is unaffected by thepresence of 5 mM EDTA, showing that Ca²⁺ is not require for 3G4 bindingto anionic phospholipids. 3G4 did not bind to ELISA plates that had beencoated with phospholipids but then washed with 0.2% Tween 20 in saline,confirming that the binding was to the absorbed phospholipid.

[1283] The epitope recognized by 3G4 appears to lie within thephosphoglycerol core of the anionic phospholipids, which is the same inphospholipids from all mammalian species. The antibody thus reacts withboth mouse and human phospholipids, which is important for pre-clinicaland clinical development. 3G4 is more specific for anionic phospholipidsthan the natural ligand, annexin V. Unlike 3G4, annexin V also bindsstrongly to neutral phospholipids in physiological concentrations ofCa²⁺.

[1284] The specificity of 3G4 for anionic phospholipids was confirmed byassays in which liposomes formed from different phospholipids were usedto compete for 3G4 binding to immobilized PS. Liposomes were preparedfrom solutions of 5 mg of a single phospholipid in chloroform. Thesolutions were dried under nitrogen to form a thin layer in around-bottomed glass flask. Ten ml of Tris buffer (0.1 M, pH 7.4) werethen added and the flask was sonicated five times for 2 min. The 3G4antibody (0.1 μg/ml) was added to either buffer or differentphospholipid liposomes and pre-incubated for 30 minutes at roomtemperature. The mixture was added to PS-coated plates (after standardblocking), incubated for 1 hour, washed and the secondary antibodyadded. After 1 hour, the plates were washed and developed for 5 minutesusing OPD.

[1285] As shown in Example IV, 3G4 binds to PS, PA, PI, PG and CL whenimmobilized and binds to immobilized PE to a lesser degree, but does notbind to immobilized PC. The ability of 3G4 to bind to immobilized PS inthe presence or absence of the different liposomes is shown in FIG. 20.Results from these liposome competition studies show that binding of 3G4to PS adsorbed to ELISA plates was blocked by liposomes prepared fromPS, PA, PI, PG and CL, but that liposomes prepared from PE and PC didnot result in a detectable reduction in 3G4 binding (FIG. 20). Also, SMliposomes were not inhibitory.

[1286] B. Inhibition of Cell Proliferation

[1287] 3G4 binds to activated, dividing, injured, apoptotic andmalignant cells that externalize PS and other anionic phospholipids. The3G4 antibody inhibits the proliferation of endothelial cells in vitro,and shows a marked selective inhibition of dividing endothelial cells asopposed to quiescent cells.

[1288] The effect of the anti-PS antibodies 3G4, 9D2, 3B10, 1B9, 2G7,7C5 and 3SB on the growth of bEnd.3 cells in vitro was determined.bEnd.3 cells (10,000/well) were seeded in 48 well plates and allowed toattach. 20% DMEM alone (control) or 20% DMEM containing the antibodies(20 μg to 40 μg total IgG per well) was added 4 hours after seeding.Each clone was tested on two separate plates in triplicates. Cells weredetached 48 and 96 hours later, the cell count was determined in eachwell and the average cell number per treatment was calculated.

[1289] The 3G4 and 9D2 antibodies were particularly effective, followedby 3SB and 3B 10, with 1B9, 2G7 and 7C5 having less inhibitory effects.Each of the antibodies show a selective inhibition of dividing(subconfluent) endothelial cells as opposed to quiescent (confluent)cells. In comparative studies, 3G4 showed the greatest inhibitoryeffect, followed by 9D2, each of which were more inhibitory than 3SB(FIG. 16).

[1290] C. Anti-Tumor Effects

[1291] 3G4 binds to the surface of tumor vascular endothelial cells invivo. When injected intravenously into mice bearing various tumors, 3G4specifically and consistently localized to the tumor, but not to normalorgans. Staining was observed on tumor vascular endothelium (FIG. 22),necrotic areas and individual malignant cells. There are multiplebinding sites for 3G4 in tumors, which allows simultaneous targeting ofboth tumor endothelial and tumor cells.

[1292] 3G4 suppresses angiogenesis and tumor growth in vivo and shows nodetectable organ toxicity in tumor-bearing mice. In initial studies, 3G4has shown impressive anti-tumor effects in syngeneic and xenogeneictumor models, wherein the antibody causes tumor vascular injury,decrease in vascularity and tumor necrosis (Example XI). Regressions ofestablished tumors have been observed in 30% to 50% of the animalstreated.

[1293] Representative anti-angiogenic and vascular targeting effects ofthe 3G4 antibody are shown in FIG. 17A and FIG. 17B, respectively.Analyses of tumor sections from nude mice bearing MDA-MB-231 orthotopictumors treated with 3G4 revealed anti-angiogenic effects in all treatedtumors. FIG. 17A shows representative images of tumors from mice treatedwith 3G4 as opposed to control antibodies. The control tumor shows nosigns of necrosis and is highly vascularized, as demonstrated by thepan-endothelial cell marker, CD31, detected on tumor blood vessels (FIG.17A, left panel). In contrast, tumors from the mice treated with 3G4have 80 to 90% necrosis and almost complete disappearance ofCD31-positive structures, indicating that the treatment produced asubstantial anti-angiogenic effect (FIG. 17A, right panel).

[1294] Another component of the anti-cancer activity of 3G4 is theinduction of tumor vascular damage. This is illustrated in FIG. 17B,which provides representative images of H&E stained tumors derived fromthe same controlled study. The blood vessels in the control tumors arewell perfused, morphologically intact and surrounded by viable dividingtumor cells (FIG. 17B, left panel). In contrast, the blood vessels inthe 3G4-treated animals are frequently observed to have a disintegratingendothelial layer and are blocked by the detached endothelial cells and,likely, by host cells that are attracted to the denuded vessels (FIG.17B, right panel). The representative vessel in the 3G4-treated tumorclearly shows loss of function, as indicated by the pre-necrotic layerof surrounding tumor cells (FIG. 17B, right panel).

[1295] In summary, the histological examination following the treatmentof orthotopic MDA-MB-231 tumors using 3G4 shows: 1) disintegration ofvascular endothelium in about 50% of vessels in the tumor; 2) attachmentof leukocytes to tumor endothelium and infiltration of mononuclear cellsinto the tumor interstitium; 3) occlusion of tumor vessels by plateletaggregates and red cells; 4) a 70% reduction in microvascular density intumors from 3G4 treated vs. untreated mice; and 5) central necrosis ofthe tumors, with survival of a peripheral rim of tumor cells, typical ofa VTA. Thus, a primary anti-tumor action of the 3G4 antibody is exertedthrough effects on tumor vasculature. Other mechanisms, particularlyantibody-dependent cellular cytotoxicity directed against the tumorcells themselves, likely contributes to the anti-tumor effect. This isimportant, and may permit killing of more tumor cells, including thosein the peripheral rim.

[1296] In follow-up studies, the effect of 3G4 on tumor growth has beenexamined in other murine models, including syngeneic (mouse Meth Afibrosarcoma), subcutaneous xenografts (L540 human Hodgkin's lymphoma)and orthotopic tumors (human MDA-MB-231 breast cancer and humanMDA-MB-435 breast cancer). Treatment of mice with 3G4 antibody resultedin 90%, 65% and 50% and 70% growth retardation of these tumors,respectively. Both small (0.1 cm diameter) and well-established (0.3 cmdiameter, 200 mm³) tumors were inhibited alike. Anti-PS treatmentinduced long-term complete remissions in 50% of Meth A-bearing mice and30% of mice with MBA-MD-231 tumors. 3G4 has the highest inhibitoryeffect in immunocompetent mice. The orthotopic models of human breasttumors (MDA-MB-231 and MDA-MB-435), in which human breast tumors aregrown in the mammary fat pads of mice, are important as these arepractical and realistic models of human breast cancer growing within thebreast of humans.

[1297] D. Safety Profile

[1298] The 3G4 antibody is different to anti-phospholipid antibodiesdescribed in the literature. Typically, anti-phospholipid antibodies areregarded as pathogenic antibodies that interfere with the coagulationcascade. They inhibit coagulation reactions in vitro and causethrombosis in vivo. In contrast, 3G4, 9D2 and like antibodies aretherapeutic antibodies without pathogenic effects.

[1299] 1. Coagulation

[1300] An important aspect of the 3G4, 9D2 and like antibodies stemsfrom the inventors' realization that desirable antibodies shouldpreferably be selected using a screen to identify antibodies that bindto PS-coated plates as strongly in the presence of serum as in theabsence of serum. This new development provides the ability to identifyand exclude antibodies that recognize complexes of PS and serumproteins, as such complexes are believed to be the cause of, or animportant factor in, anti-phospholipid syndrome and associatedpathologies.

[1301] In studies of blood coagulation in vitro, a weak inhibition ofTissue Factor (TF)-induced coagulation was observed using high doses of3G4 antibody. In other studies using lower doses, recalcified plasmafrom 3G4 treated mice coagulated at the same rate as did recalcifiedplasma from BBG3 treated mice in the presence of tissue factor. Also,the addition of 100 μg/ml of 3G4 to cells plus tissue factor in vitrodid not affect the generation rate of coagulation Factor Xa in proplex(extrinsic coagulation pathway).

[1302] Despite the weak inhibition of TF-induced coagulation using highantibody levels in vitro, the 3G4 antibody has been tested in vivo anddoes not cause thrombotic complications in normal or tumor-bearing mice(e.g., see Example XI). The 3G4 antibody has also been tested in monkeysin vivo and no significant side effects have been observed.

[1303] 2. Other Indicators of Low or No Toxicity

[1304] The first evidence that 3G4 has no or low toxicity in mice camefrom the finding that 3G4 grows as a hybridoma in mice without evidenceof toxicity. Also, when 1 mg of purified 3G4 was injectedintraperitoneally, no toxicity was observed.

[1305] Systematic in vivo studies have now been conducted in whichgroups of three 8 week old BALB/c mice were injected IP with 100 μg ofpurified 3G4 or with an isotype-matched control IgG₃ (BBG3) three timesa week for 2 to 3 weeks. No physical signs of toxicity have beenobserved, and no histopathological signs of organ toxicity ormorphological abnormalities have been detected in sections of majororgans removed from 3G4-treated mice. The following parameters werespecifically examined.

[1306] In terms of bodyweight, 3G4-treated mice gained weight at thesame rate as BBG3 treated mice. No weight loss was observed in theearlier studies. There were no physical signs of toxicity, e.g. hairloss, loss of appetite, etc. There are no changes in blood cell counts,including red cells, platelets, white cells, absolute lymphocyte countsor absolute neutrophil counts. To analyze bone marrow cellularity,paraffin sections of bone marrow derived from 3G4 or BBG3-treated mice(six injections, 100 μg) were examined for total cellularity andcellular composition. Marrows in the treated animals were essentiallycompletely cellular (as would be expected for a young mammal).Erythroid, granulocytic, lymphocytic progenitors and megakaryocytes werepresent in normal numbers.

[1307] In summary, no instance of toxicity has been observed in morethan 200 mice treated with high doses of 3G4 (0.1 mg) three times a weekfor 2-3 weeks. Even when doses as high as 2 mg were given, no signs oftoxicity were seen. Mice retain normal physical signs, bone marrowcellularity, white blood cell counts, histology and coagulationfunctions.

[1308] The 3G4 antibody has also been administered to monkeys in safetystudies and no side effects have been observed.

[1309] Blood clearance kinetic studies have also been conducted in mice.3G4 was radioiodinated using the Bolton Hunter reagent and was injectedintravenously into mice (25 g). Samples of blood were removed via thetail vein at various later time points. The blood clearance rate of 3G4was typical of a mouse IgG in the mouse. The half-life in the α-phase ofclearance was 3 hours while that in the β-phase was 5 days. Volume ofdistribution was normal (100 ml/kg). These studies indicate that 3G4does not interact with normal host tissues, leading to its acceleratedclearance.

[1310] E. Anti-Viral Effects

[1311] The 3G4 antibody also exerts significant anti-viral effects. Asshown in seen in Example XIII, the treatment of RSV-infected cells with3G4 was superior to the effect observed using 3SB. These resultstherefore highlight another advantage of the 3G4 antibody over theprominent anti-PS antibody in the literature, 3 SB (Rote et al. (1993).

[1312] The 3G4 antibody is also shown to be very effective in inhibitingCMV, both in vitro (Example XII) and in enhancing the survival of miceinfected with mCMV in vivo (Example XXI). In addition, the 3G4 antibodyis further shown to inhibit Pichinde virus infection, the infectiousagent of Lassa fever (Example XXIV). The cell surface PS exposure hereinshown to follow viral infection, and the ability of the 3G4 antibody tobind to cells infected with Vaccinia virus (Example XXIII), shows thatthe 3G4 antibody has enormous potential as a broad spectrum anti-viralagent.

EXAMPLE XIX 3G4 Antibody, CDR Sequences and Chimera

[1313] The 3G4 antibody thus possesses the combined properties of ananti-angiogenic, anti-tumor vascular and anti-viral agent. Theinhibitory activities of 3G4 on cell division, angiogenesis, tumorgrowth and viral infectivity, taken together with lack of apparenttoxicity, show broad therapeutic indications for this antibody,including in the treatment of angiogenic disorders, cancer, diabetes andviral infections.

[1314] Antibodies recognizing substantially the same epitope as the 3G4antibody can be generated for use in one or more of the anti-angiogenic,anti-tumor vascular and anti-viral therapies, e.g., by immunization andconfirmed by antibody competition studies. Antibodies that bind toessentially the same epitope as the 3G4 antibody can also be generatedfrom a knowledge of the 3G4 antibody sequences provided herein. Thepresent example provides the sequences of the complementaritydetermining regions (CDRs) of the 3G4 antibody and the use of thesequence information.

[1315] A. 3G4 Antibody Sequences

[1316] The original sequences of the antibody variable regions wereobtained by RACE from the hybridoma that produces the 3G4 antibody andthe sequences verified. The nucleic acid and amino acid sequences of thevariable region of the heavy chain (Vh) of the 3G4 antibody CDR1-3 arerepresented by SEQ ID NO:1 and SEQ ID NO:2, respectively.

[1317] SEQ ID NO:1 and SEQ ID NO:2 include part of the mouse leadersequence and constant chain sequences, as shown in FIG. 18A. The leadersequence is represented by amino acids 1 through 19 of SEQ ID NO:2, andthe mature protein begins as shown by the arrow in FIG. 18A. Sufficientcomplementarity determining region sequence information is included bythe sequence of the mature protein up to the sequence portion concludingVSS, after which the amino acids are not essential for antigen binding.As such, the BstEII site in the nucleic acid sequence can be used as aconvenient site to prepare a functional mouse variable region, e.g., foruse in grafting onto a human constant region (FIG. 18A).

[1318] In practice, the 3G4-2BVH sequence has been grafted onto a humanγ1 constant region at the BstEII site using a Lonza pEE vector. Theresultant product contains the mouse leader sequence and its VH isjoined to the human CH1 sequence in the manner shown in FIG. 18A,wherein ASTLGPSVFPLAPSSKSTSG (SEQ ID NO:7) represents the first part ofthe human CHI sequence.

[1319] The nucleic acid and amino acid sequences of the variable regionof the light chain (Vκ) of the 3G4 antibody CDR1-3 are represented bySEQ ID NO:3 and SEQ ID NO:4, respectively. SEQ ID NO:3 and SEQ ID NO:4again include part of the mouse leader sequence and constant chainsequences, as shown in FIG. 18B. The leader sequence is amino acids 1through 22 of SEQ ID NO:4, and the mature protein begins as shown by thearrow in FIG. 18B. Sufficient complementarity determining regionsequence information is included by the sequence of the mature proteinup to the sequence portion concluding TVF, after which the amino acidsare not essential for antigen binding. As such, the BbsI site in thenucleic acid sequence can be used as a convenient site to prepare afunctional mouse variable region, e.g., for use in grafting onto a humanconstant region (FIG. 18B).

[1320] In practice, the 3G4-2BVL sequence has been grafted onto a humanK constant region at the BbsI site using a Lonza pEE vector. Theresultant product contains the mouse leader sequence and its VL isjoined within the human CL1 sequence in the manner shown in FIG. 18B,wherein IFPPSDEQLKSGTAS (SEQ ID NO:8) represents the first part of thehuman K constant region sequence.

[1321] B. Generation and Characterization of 3G4 Chimeric Antibody

[1322] The chimeric construct containing the murine complementaritydetermining regions and the human constant regions has been produced(ch3G4) and shown to behave essentially the same as the original murineantibody.

[1323] The murine 3G4 antibody was converted into a human-mouse chimericantibody (Avanir (Xenerex) Biosciences San Diego, Calif.). The murine VHwas cloned and grafted onto the human γ₁ constant region at the BstEIIsite of the Lonza 2BVH vector. The murine V_(κ) was cloned and graftedonto the human K constant region at the BbsI site of the Lonza 2BVLvector. The sequences were verified. The entire construct was expressediii CHO cells and purified.

[1324] The resultant ch3G4 bound at least as did well as the murine 3G4to phospholipid-coated ELISA plates. The in vitro binding profile ofchimeric 3G4 to the panel of phospholipids is shown in FIG. 21, whereinbinding to PS, PA, CL, PI and PG is shown to be similar. The binding wasantigen-specific since no binding was observed with control antibodiesof irrelevant specificity. In certain studies, an apparently greaterbinding of chimeric 3G4 vs. the 3G4 antibody was observed; this may bedue to superior binding of the secondary antibody.

[1325] In vivo, ch3G4 localizes to tumor vascular endothelium and exertsanti-tumor effects. The anti-tumor effects of ch3G4 in MDA-MB-435 humanbreast cancer cells growing in mice is described in Example XI and shownin FIG. 8G. Treatment of mice with MDA-MB-435 tumors using the chimericantibody effectively retarded tumor growth as opposed to control.

[1326] Localization of ch3G4 was examined in MDA-MB-435 human breastcancer cells growing in mice. Mice were injected intravenously withbiotinylated ch3G4 or control IgG of irrelevant specificity. One hourlater, the mice were exsanguinated, and their tumors were removed andfrozen sections were cut. Biotinylated reagents were first incubatedwith streptavidin-Cy3 conjugate, washed in PBS, then incubated with MECA32 antibody followed by FITC-tagged secondary antibody. Single images,taken with appropriate filters for Cy3 (red) and FITC (green)fluorescence respectively, were captured by digital camera andtransferred to a computer. Converged images demonstrating yellow color(a product of merged green and red fluorescence) were superimposed withthe aid of Metaview software.

[1327] In this double staining method, the biotinylated proteins and thevascular endothelium are labeled by red and green. Where thebiotinylated proteins are bound to the endothelium, the converged imageappears yellow. As shown in FIG. 22, biotinylated ch3G4 binds to thetumor vascular endothelium, because the staining patterns converges withthat of MECA 32.

EXAMPLE XX 3G4 Antibody in Combination Therapy with Docetaxel

[1328] The present example concerns combination therapies for tumortreatment using the 3G4 antibody and the chemotherapeutic drug,docetaxel. These agents are designed to attack tumor vasculatureendothelial cell and tumor cell compartments, leading to synergistictreatment with lower toxicity. The results showed that this combinationtherapy did indeed significantly enhanced treatment efficacy.

[1329] A. Fc Domain-Mediated Anti-Tumor Effects

[1330] The 3G4 antibody was tested for inhibitory effects on tumor cellsin vitro. No direct inhibitory effect on tumor cells was observed.Therefore, it is likely that the anti-tumor effects of the 3G4 antibodyinclude Fe domain-mediated augmentation of immune effector functions,such as antibody mediated phagocytosis, ADCC, CDC and stimulation ofcytokine production, or these mechanisms combined.

[1331] The effects of 3G4 on the phagocytosis of PS-positive cells bymacrophages have been evaluated. Fluorescent tumor cells were treatedwith H₂O₂ to induce PS exposure. Treated and untreated cells were thenharvested and contacted with the 3G4 antibody or a control antibody(BBG). Mouse bone marrow macrophages were then added, and the ability ofthe macrophages to phagocytose the fluorescent tumor cells was analyzedusing a fluorescent microscope.

[1332] It was determined that 3G4 could increase the phagocytosis ofPS-positive cells by macrophages by more than three fold (FIG. 23). Thisfinding supports the inventors' reasoning that the Fc domain of the 3G4antibody contributes to the anti-tumor effects of the antibody. That is,the Fc domain activates host immune effector functions, which then exertanti-tumor effects. The 3G4 antibody should therefore enhance the lyticactivity of NK cells, leading to more effective ADCC.

[1333] B. Docetaxel Induces PS Exposure on Endothelial Cells

[1334] The induction of PS exposure on endothelial cells by subclinicalconcentrations of docetaxel was examined in vitro by FACS analysis.Human umbilical vein endothelial cells (HUVEC) and human microvesselendothelial cells (HMVEC) were treated with 10 nM of docetaxel for 24hrs and examined by FACS. Both treated HUVEC and HMVEC showedsignificant increase in 3G4 binding as compared to untreated cells (FIG.24A and FIG. 24B, respectively). Docetaxel incubations for 48 and 72 hrswere also conducted.

[1335] C. Docetaxel Induces PS Exposure on Tumor Cells

[1336] The in vitro induction of PS exposure by subclinicalconcentrations of docetaxel was also examined by FACS analysis using apanel of tumor cell lines. Mouse lewis lung carcinoma 3LL, mouse coloncarcinoma Colo26 and human breast cancer MDA-MB-435 cells were treatedwith 10 nM of docetaxel for 24 hrs and examined by FACS. All tumor celllines tested showed significant increase in 3G4 binding as compared withuntreated cells (FIG. 25A, FIG. 25B and FIG. 25C, respectively).Docetaxel incubations for 48 and 72 hrs were also conducted. Mousemelanoma B 16 and mouse firbrosarcoma Meth A tumor cell lines werefurther examined and also showed significant increase in 3G4 binding ascompared with untreated cells.

[1337] Human breast cancer MDA-MB-231 cells were treated with 10 nM ofdocetaxel for 24 hrs and incubated with either the chimeric 3G4 antibody(ch3G4) or control, human IgG and analyzed by FACS. These results showthat the significant increase in antibody binding is antigen-specificand that the chimeric antibody behaves like the parent 3G4 antibody(FIG. 26).

[1338] D. Synergistic Tumor Treatment with 3G4 and Docetaxel

[1339] The inventors have thus shown that the treatment of endothelialcells and tumor cells with docetaxel at subclinical concentrationsignificantly increases 3G4 binding. They have also shown that the 3G4antibody facilitates macrophage-mediated phagocytosis of tumor cells onwhich PS is exposed at the surface. The increased 3G4 binding mediatedby docetaxel should therefore augment the phagocytosis of tumor cellsand other anti-tumor effects mediated by the Fc domain of the 3G4antibody, such as increasing the lytic activity of NK cells, leading tomore effective ADCC. Studies of others have also shown that treatment ofbreast cancer patients with docetaxel leads to an increase in serumIFN-γ, IL-2, IL-6 and GM-CSF cytokine levels and enhancement of NK andLAK cell activity (Tsavaris et al., 2002).

[1340] The anti-tumor effect of the combined therapy of 3G4 withdocetaxel was therefore examined in an orthotopic model in SCID micebearing human MDA-MB-435 breast carcinoma. Mice bearing orthotopicMDA-MB-435 human breast tumor were treated i.p. with 3G4 alone (100μg/dose), docetaxel alone (11 mg/kg), or 3G4 in combination withdocetaxel (100 μg/dose and 10 mg/kg, respectively), for three weeks,with administration 3 times a week. Treatment started 6 days after tumorcell implantation.

[1341] These studies showed that the combined therapy of 3G4 plusdocetaxel resulted in growth inhibition of 90%. Growth inhibition of 3G4plus docetaxel was significantly superior to 3G4 alone (p<0.005) anddocetaxel alone (p<0.01).

[1342] E. 3G4-Targeting of Apoptotic Tumor Cells to FcγR on DendriticCells

[1343] Tumors from mice treated with 3G4 plus docetaxel also containedunusual amount of lymphocytes, as compared to control tumors. Althoughthis phenomenon could represent typical chemoattraction of immune cellsby disintegrating tumor cells, it could also reflect activation of theimmune system by 3G4 mediated through Fc binding to FcγR on immuneeffector cells.

[1344] To characterize the effects of 3G4 and docetaxel administrationon the intratumoral immune cell infiltrate, the types of cells presentin these infiltrates can be identified by immunostaining of frozensections and/or paraffin sections of tumor tissues using antibodiesdirected against specific markers of macrophages, neutrophils,granulocytes, NK cells and activated lymphocytes (Pharmingen, San Diego,Calif.). The extent, phenotype, and activation status of this infiltratecan be graded. Cytokine production by infiltrating immune cells,including IL-2 and INF, can also be analyzed via immunohistochemicaltechniques. Serum cytokine levels can be evaluated by ELISA andintracellular staining can be used to identify the specific cellularcompartments responsible for cytokine production. The effects ofinfiltrating immune cells on tumor cell proliferation and apoptosis canthus be systematically evaluated.

[1345] In light of the foregoing data, the inventors further contemplatemethods enhancing the potency of immunotherapy of breast cancer by3G4-mediated targeting of apoptotic tumor cells to the Fc gamma receptor(Fc(γ)R) on dendritic cells. Efficient antigen presentation, whichinduces effective cellular and humoral immune responses, is importantfor the development of tumor vaccines and immunotherapies. Dendriticcells (DC) are the most potent antigen-presenting cells (APC) that primecytotoxic T lymphocytes against tumor-associated antigens. Improvementof tumor antigen presentation by dendritic cells (DCs) should lead todevelop more potent tumor vaccines.

[1346] Antigenic presentation by Fc(γ)R receptor-mediatedinternalization of DCs can be enhanced up to 1,000-fold compared withfluid phase antigen pinocytosis. Apoptotic tumor cells (ATC) are anexcellent source of antigens for dendritic cell loading because multipletumor specific antigens (both known and unknown) can be efficientlypresented to naive T cells, making the occurrence of immune escapevariants less likely due to the lock of certain epitopes. In animalstudies, DCs pulsed with ATCs have been shown to produce potentanti-tumor immunity in vitro and in vivo. However, recent data hasdemonstrated that ATCs alone were somewhat inefficient for activatinganti-tumor immunity, possibly because of their insufficient uptake andinability to induce DC maturation.

[1347] Recent studies have also demonstrated that ATC-immune complex,formed by binding of anti-tumor antibody to apoptotic tumor cells, canbe targeted to Fc(γ)R on DC. Compared with ATCs alone, ATC-immunecomplexes were more efficiently internalized by DC, more efficient ininducing DC activation and maturation, and more importantly, ATC-immunecomplexes can significantly enhance both MHC I and II-restricted antigenpresentation, therefore induce potent anti-tumor T helper and CTLimmunity.

[1348] The inventors therefore envision using the anti-PS antibodies ofthe present invention to enhance both hormonal and cellular anti-tumorimmunity, and boost the efficacy of ATC based DC tumor vaccines. As PSis a universal and the most abundant specific marker of apoptotic tumorcells, the panel of antibodies of the invention, particularly 3G4, canbind to PS on ATCs. The inventors have already demonstrated that 3G4 canenhance DC uptake of apoptotic tumor cells by 300% through Fc(γ)Rmediated internalization of 3G4-ATC complexes. By enhancing the uptakeof ATC by DC mediated through Fc(γ)R, it is therefore reasoned that 3G4and like antibodies can greatly enhance both MHC I and II restrictedantigen presentation, induce both potent hormonal and cellularanti-tumor immunity, and boost the efficacy of ATC based DC tumorvaccines. This can be demonstrated by establishing the efficacy of DCloaded with 3G4-ATC immune complexes in the induction of T h1, CTL andantibody response in vivo, and by determining the potency of anti-tumorimmunity induced by immunization of DC loaded with 3G4-ATC immunecomplexes in vivo.

EXAMPLE XXI

[1349] Anti-PS Antibodies Treat CMV Infections In Vivo

[1350] Following the anti-viral effects against CMV in vitro shown inExample XII, the present example demonstrates the enhanced survival ofmice infected with the murine version of the CMV virus, mCMV.

[1351] Balb/C mice (6 week old, five mice per group) were infected i.p.with 5×10⁵ pfu of mCMV RVG102. The mice were treated i.p. on day 1 withthe 3G4 antibody (1 mg/mouse), or the human-mouse chimeric antibody,ch3G4 described above (1 mg/mouse). Untreated mice served as thecontrol. The mice were treated every four days thereafter with 0.5mg/mouse of antibody or chimeric antibody until day 26. The mice weremonitored for survival past 90 days post infection.

[1352] Treatment with both the parent and chimeric forms of the 3G4antibody resulted in increased survival of the mCMV-infected mice. Micetreated with 3G4 or ch3G4 had 100% and 80% survival, respectively, ascompared to untreated mice, wherein only 25% of the mice survived theinfection (FIG. 27).

EXAMPLE XXII

[1353] PE-Binding Peptide Derivative Treats CMV Infection In Vivo

[1354] In addition to the in vitro anti-viral effects against CMV shownin Example XVII, this example demonstrates that the duramycin-biotinderivative, DLB increased survival of mice infected with mCMV.

[1355] Balb/C mice (6 week old, five mice per group) were infected i.p.with 5×10⁵ pfu of mCMV RVG102. The mice were treated i.p. on day 1 andevery four days with 20 μg/mouse of the duramycin derivative, DLB.Untreated mice served as the control. The mice were monitored forsurvival past 90 days post infection.

[1356] Treatment with the duramycin-biotin derivative, DLB enhancedsurvival of the mCMV-infected mice. Mice treated with DLB had 100%survival, as compared to untreated mice, wherein only 25% of the micesurvived the infection (FIG. 28).

EXAMPLE XXIII

[1357] Anti-PS Antibodies Bind to Virally Infected Cells

[1358] The present example shows that viral infection induces PSexposure at the cell surface and that anti-PS antibodies bind to virallyinfected cells. Cells infected with Vaccinia virus become PS-positive,as shown by increased binding of the chimeric 3G4 antibody to the cellsurface demonstrated in FACS analyses.

[1359] U937 cells were infected with trypsinized Vaccinia virus at ahigh m.o.i of 2. Briefly, Vaccinia virus was treated with an equalvolume of 0.25 mg/ml trypsin for 30 minutes at 37° C. The virus wasadded to U937 cells in a total volume of 0.5 ml. After 1.5 hr, freshmedium was added to the cells and the cells were incubated in a T25flask at 37° C. for 2 days. Uninfected cells served as the controls.

[1360] Infected and uninfected U937 cells were stained with a primaryantibody, either with the chimeric 3G4 antibody (ch3G4) or with humanIgG (HIgG) as a control. The cells were washed, blocked with normalmouse serum and then stained with the primary antibody for 45 minutes onice. After three washes, the cells were stained with a 1:400 dilution ofgoat anti-human FITC-conjugated secondary antibody and were analyzed ona FACScan.

[1361] Results from the FACS analyses show that there is a significantshift with ch3G4 on U-937 cells infected with Vaccinia virus (FIG. 29B,right (green) peak), as compared to that obtained on uninfected U937cells (FIG. 29A, right (green) peak). This study therefore shows thatinfection of cells with Vaccinia virus leads to PS exposure on the cellsurface and that the chimeric version of the anti-PS antibody, 3G4 iscapable of binding to these virally infected cells.

EXAMPLE XXIV

[1362] Anti-Viral Effects of Anti-PS Antibodies Against Pichinde Virus

[1363] In addition to the anti-viral effects against CMV and RSV, thepresent example further shows that anti-PS antibodies inhibit Pichindevirus infection in vitro. Pichinde virus is New World arenavirus, whichis non-pathogenic in man, and is used in an animal model for Lassafever.

[1364] Confluent monolayers of Vero cells were treated with the 3G4antibody or an isotype-matched control antibody, GV39G, after infectionwith Pichinde virus at a low m.o.i. of 0.01 pfu/cell. Briefly, the cellswere incubated with virus in a total volume of 1 ml per well at 37° C.for 90 minutes. During the infection, the plates were gently rockedevery 30 minutes. Following the infection, the cell supernatant wasremoved and DMEM/10% FBS/pen-strep was added to each well (2 ml perwell). On day 2, the cells were harvested with trypsin and allowed toadhere to Biocoat chamber slides. They were fixed and stained withpolyclonal rabbit anti-PIC serum followed by a biotin-conjugated goatanti-rabbit secondary (secondary antibody alone produced no staining, asshown in FIG. 30C). The number of infected cells per field of 100 cellswas counted.

[1365] In cells treated with 3G4, the virus is restricted to singlecells that stain a dark red, numbering about one in about a hundredcells (FIG. 30A). These are probably the cells that were originallyinfected by the virus, as was seen with CMV (Example XII). However, incells treated with the control, GV39G antibody, the virus has spread andinfected all the cells (FIG. 30B).

[1366] This pattern of inhibition of viral replication is similar tothat observed when 3G4 was used to treat CMV-infected human fibroblasts.Thus, the anti-PS antibody, 3G4 effectively prevents the spread ofPichinde virus from cell to cell, as quantified in FIG. 30D.

EXAMPLE XXV Tumor Treatment Using PE-Binding Peptide Derivative

[1367] Further to the anti-viral effects of duramycin derivatives, bothin vitro and in vivo, the present example demonstrates the localizationof duramycin derivatives to tumor vasculature and associated anti-tumoreffects.

[1368] A. Tumor Treatment with Duramycin-HuIgG Conjugate

[1369] Human IgG (HIgG) was first purified as described in Example XV.Purified HIgG was linked to duramycin using the SIAB linker, and theresultant (D-SIAB)_(n)HIgG conjugate purified.

[1370] Mouse fibrosarcoma cell-line MethA was grown, harvested at logphase and resuspended in DPBS. Approximately 10⁶ MethA tumor cells wereinjected subcutaneously in the middle dorsum of 6-8 week old BALB/c malemice. 5 days after implantation, the mice were randomly separated intotwo groups (n=15). From day 10, one group received 150 μgDuramycin-HuIgG conjugate by intraperitoneal injection for consecutive 2weeks. The other group received the same amount of HuIgG as a control.Tumor volumes were measured twice a week and were calculated using theformula 1/2ab², (where “a” is the long axis and “b” the short axis ofthe tumor). Mice were sacrificed when the tumors reached a size ofapproximately 1400 mm³.

[1371] The duramycin-HIgG conjugate inhibited MethA tumor growth inBALB/c mice at the dose of 150 μg/day, as compared to the human IgGcontrol (FIG. 31).

[1372] B. Duramycin-HuIgG Conjugate Localizes to Tumor Vasctilature

[1373] Using the same MethA mouse tumor model as above, when the tumorsize reach 500 mm³, 100 μg (D-SIAB)_(n)HIgG in 100 μl PBS was injectedthrough the tail vein. The same amount of human IgG was injected as acontrol. After 4 hours, mice was euthanized and perfused with normalsaline for 5 minutes and 1% paraformadehyde for 10 minutes. The tumorand other major organs were dissected and frozen in liquid nitrogen.After embedding in OCT, tissue was cryosected in 10 μm section andplaced on silanized slides. After fixing in cold acetone for 10 minutes,slides were stained with peroxidase labeled goat anti human IgG todetect the biodistribution of duramycin-HuIgG. Meca32 and peroxidaselabeled goat anti-rat IgG were used to detect blood vasculature oftissue.

[1374] This study showed that the duramycin-HIgG conjugate localized tothe tumor vasculature in the treated animals.

EXAMPLE XXVI Biodistribution and Properties of Duramycin Conjugates

[1375] The present example demonstrates the lack of toxicity ofcell-impermeant duramycin derivatives in vitro, the biodistribution ofduramycin derivatives administered in vivo and the ability ofduramycin-antibody conjugates to increase the phagocytosis of apoptoticcells by macrophages.

[1376] A. Duramycin-Biotin Conjugates are Not Cytotoxic

[1377] The duramycin derivatives and conjugates of the invention aredesigned to minimize the non-specific toxic effects of the parentduramycin molecule. In many examples, this is achieved by linkingduramycin to a cell impermeant group (Example XV).

[1378] The biotinylated duramycin construct DLB was prepared asdescribed in Example XV. The unmodified duramycin compound and DLB weretested for cytotoxic effects on HUVEC using an MTT assay. Whilst theunmodified duramycin showed dose-dependent toxicity, DLB was non-toxic,matching the untreated control (FIG. 32).

[1379] B. Localization of Duramycin-Biotin Conjugate to Macrophages inLung

[1380] The human breast cancer cell line MDA-MB-435 was grown, harvestedat log phase, and resuspended in DPBS. Approximately 10⁷ cells wereinjected into the mammary fat pad of 6-8 week old female ethylic nudemice. 100 μg duramycin-biotin in 100 μl PBS was injected through thetail vein. After 4 hours, mice was euthanized and perfused with normalsaline for 5 minutes and 1% paraformadehyde for 10 minutes. Majororgans, including heart, lung, liver, kidney, brain, intestine, testesand spleen were dissected and frozen in liquid nitrogen. After embeddingin OCT, tissue was cryosected in 10 μm sections and placed on silanizedslides. After fixing in cold acetone for 10 minutes, slides were stainedwith Cy3 labeled streptavidin to detect the biodistribution of theduramycin-biotin construct. Meca32 and FITC labeled goat anti rat IgGwere used to detect blood vasculature of tissue.

[1381] The intravenous injection of the duramycin-biotin conjugate intonude mice bearing MDA-MB-435 tumors resulted in the deposition of drugin the tumor cells, renal tubules and in the macrophages in the lung.There was minimal deposition in liver and no detectable distribution inbrain, intestine, testes. The localization to macrophages in the lungcan be exploited in the anti-viral embodiments of the invention.

[1382] C. Duramycin-Antibody Conjugate Enhances Phagocytosis ofApoptotic Cells

[1383] The ability of a duramycin-antibody conjugate (duramycin-C44,DuC44) to increase the phagocytosis of apoptotic cells was nextinvestigated.

[1384] Macrophages were isolated and cultured from mouse bone marrow.The medium used for the isolation, culture, and stimulation of BMmacrophages was DMEM containing 2 mM glutamine, 0.37% (w/v) NaHCO₃, 10%(v/v) heat-inactivated FCS, and 0.5 ng/ml mouse GM-CSF. Bone marrowcells were flushed asceptically from the dissected femurs with jet ofcomplete medium directed through a 25-gauge needle. The cells were thenadjusted to a density of approximately 3×10⁵ cells/ml of completemedium, and were distributed in 0.5 ml aliquots into 8 well chamberslides.

[1385] Cells were incubated for 1 hour at 37° C. in 5% CO2, in ahumidified chamber to allow macrophages to adhere and spread.Nonadherent cells were removed by adding 5 ml of warmed PBS to eachwell, resuspending nonadherent cells by moderately tapping the plate,and flicking the slides to discard the nonadherent cells. This washingwas performed a total of three times. The cells were maintained at 37°C. under a 7.5% (v/v) CO₂ atmosphere for 5 days. The complete medium waschanged every other day until the cells were used.

[1386] The following method was used to label HL-60 target cells with afluorescent cell tracer. A 10 mM CFDA SE stock solution was preparedimmediately prior to use by dissolving the contents of one vial dye in90 μL of the DMSO and diluting in PBS to 10 μM. Centrifugation was usedto obtain a HL-60 cell pellet and the supernatant aspirated. The cellswere resuspend in CFDA/PBS and incubated at 37° C. for 15 minutes. Thesamples was centrifuged and the supernatant aspirated. The cells wereresuspend in media and incubated for another 30 minutes. The cellviability and fluorescence were confirmed to be over 95%.

[1387] In this phagocytosis assay, labeled HL-60 cells were exposed toUV 254 nm for 5 minutes and incubated at 37° C. for one hour to induceapoptosis. 104 apoptotic HL-60 cells were incubated with macrophages forone hour. Duramycin-C44 conjugate was included at the concentration of10 μg/ml. The same concentration of mouse antibody BBG3 was used as anegative control, and the 3G4 antibody was also included for comparison.Iloechst 33342 was added in media in the last 45 minutes at theconcentration of 10 μg/ml.

[1388] Slides were washed with PBS 3 times, and fixed in 4%paraformadehyde for 15 minutes. The slides were stained with ratanti-mouse CD11 antibody (CD11 is a macrophage marker), diluted in 0.2%gelatin for one hour, washed and stained with Texas red labeled goatanti-rat secondary antibody.

[1389] The cells were analyzed under the fluorescence microscope.Macrophages are identified as red cells, due to the CD11 marker.Macrophages that have phagocytosed apoptotic cells are identified asgreen cells, due to the fluorescent tracer in the target cells. Red andgreen cells are counted and the phagocytosis is quantified as thepercent phagocytes positive for uptake.

[1390] This study shows that the duramycin-antibody conjugate, DuC44enhanced phagocytosis of apoptotic HL-60 cells by macrophages (FIG. 33).Thus, the duramycin portion is binding to the surface of the apoptoticcells, permitting the protruding antibody portion of the conjugate to berecognized by the macrophages. The duramycin-antibody conjugate thusfunctioned similarly to the 3G4 antibody. As expected, an (Fab)₂fragment of the 3G4 antibody, lacking the Fc region, did not inducephagocytosis above control levels.

[1391] As the earlier study showed duramycin-biotin conjugates tolocalize to macrophages in the lung following administration in vivo,the stimulation of macrophage-mediated phagocytosis of apoptotic cellsshown in the present study has important implications for thetherapeutic uses of the present invention, such as in treating pulmonaryviral infections.

[1392] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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1 9 1 519 DNA Mus musculus 1 atgggatgga cctggatctt tattttaatc ctgtcagtaactacaggtgt ccactctgag 60 gtccagctgc agcagtctgg acctgagctg gagaagcctggcgcttcagt gaagctatcc 120 tgcaaggctt ctggttactc attcactggc tacaacatgaactgggtgaa acagagccat 180 ggaaagagcc ttgaatggat tggacatatt gatccttactatggtgatac ttcctacaac 240 cagaagttca ggggcaaggc cacattgact gtagacaaatcctccagcac agcctacatg 300 cagctcaaga gcctgacatc tgaggactct gcagtctattactgtgtaaa ggggggttac 360 tacgggcact ggtacttcga tgtctggggc gcagggaccacggtcaccgt ctcctcagct 420 acaacaacag ccccatctgt ctatcccttg gtcccgggcggatcccccgg gctgcaggaa 480 ttcgatatca agcttatcga taccgtcgac ctcgagggg 5192 152 PRT Mus musculus 2 Met Gly Trp Thr Trp Ile Phe Ile Leu Ile Leu SerVal Thr Thr Gly 1 5 10 15 Val His Ser Glu Val Gln Leu Gln Gln Ser GlyPro Glu Leu Glu Lys 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys AlaSer Gly Tyr Ser Phe 35 40 45 Thr Gly Tyr Asn Met Asn Trp Val Lys Gln SerHis Gly Lys Ser Leu 50 55 60 Glu Trp Ile Gly His Ile Asp Pro Tyr Tyr GlyAsp Thr Ser Tyr Asn 65 70 75 80 Gln Lys Phe Arg Gly Lys Ala Thr Leu ThrVal Asp Lys Ser Ser Ser 85 90 95 Thr Ala Tyr Met Gln Leu Lys Ser Leu ThrSer Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Val Lys Gly Gly Tyr TyrGly His Trp Tyr Phe Asp Val 115 120 125 Trp Gly Ala Gly Thr Thr Val ThrVal Ser Ser Ala Thr Thr Thr Ala 130 135 140 Pro Ser Val Tyr Pro Leu ValPro 145 150 3 435 DNA Mus musculus 3 atggacatga gggctcctgc acagattttgggcttcttgt tgctcttgtt tccaggtacc 60 agatgtgaca tccagatgac ccagtctccatcctccttat ctgcctctct gggagaaaga 120 gtcagtctca cttgtcgggc aagtcaggacattggtagta gcttaaactg gcttcagcag 180 ggaccagatg gaactattaa acgcctgatctacgccacat ccagtttaga ttctggtgtc 240 cccaaaaggt tcagtggcag taggtctgggtcagattatt ctctcaccat cagcagcctt 300 gagtctgaag attttgtaga ctattactgtctacaatatg ttagttctcc tcccacgttc 360 ggtgctggga ccaagctgga gctgaaacgggctgatgctg caccaactgt cttcatcttc 420 gggcggatcc cccgg 435 4 144 PRT Musmusculus 4 Met Asp Met Arg Ala Pro Ala Gln Ile Leu Gly Phe Leu Leu LeuLeu 1 5 10 15 Phe Pro Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser ProSer Ser 20 25 30 Leu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys ArgAla Ser 35 40 45 Gln Asp Ile Gly Ser Ser Leu Asn Trp Leu Gln Gln Gly ProAsp Gly 50 55 60 Thr Ile Lys Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp SerGly Val 65 70 75 80 Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp TyrSer Leu Thr 85 90 95 Ile Ser Ser Leu Glu Ser Glu Asp Phe Val Asp Tyr TyrCys Leu Gln 100 105 110 Tyr Val Ser Ser Pro Pro Thr Phe Gly Ala Gly ThrLys Leu Glu Leu 115 120 125 Lys Arg Ala Asp Ala Ala Pro Thr Val Phe IlePhe Gly Arg Ile Pro 130 135 140 5 783 DNA ARTIFICIAL SEQUENCE SYNTHETICOLIGONUCLEOTIDE 5 gcccagccgg ccatggccga ggtgcagctg gtggagtctg ggggaggcgtggtccagcct 60 gggaggtccc tgagactctc ctgtgcagcc tctggattca ccttcagtagctatggcatg 120 cactgggtcc gccaggctcc aggcaagggg ctggagtggg tggcagttatatcatatgat 180 ggaagtaata aatactatgc agactccgtg aagggccgat tcaccatctccagagacaat 240 tccaagaaca cgctgtatct gcaaatgaac agcctgagag ctgaggacacggccgtgtat 300 tactgtgcaa gattgcatgc tcagacttgg ggccaaggta ccctggtcaccgtctcgagt 360 ggtggaggcg gttcaggcgg aggtggctct ggcggtagtg cacttcagtctgtgctgacg 420 cagccgcctt cagtgtctgc ggccccagga cagaaggtca ccatctcctgctctggaagc 480 agctccgaca tggggaatta tgcggtatcc tggtaccagc agctcccaggaacagccccc 540 aaactcctca tctatgaaaa taataagcga ccctcaggga ttcctgaccgattctctggc 600 tccaagtctg gcacctcagc caccctgggc atcactggcc tctggcctgaggacgaggcc 660 gattattact gcttagcatg ggataccagc ccgcggaatg tattcggcggagggaccaag 720 ctgaccgtcc taggtgcggc cgcacatcat catcaccatc acggggccgcagaacaaaaa 780 ctc 783 6 261 PRT ARTIFICIAL SEQUENCE POLYPEPTIDE 6 AlaGln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly 1 5 10 15Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 20 25 30Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly 35 40 45Lys Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys 50 55 60Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70 7580 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 85 9095 Thr Ala Val Tyr Tyr Cys Ala Arg Leu His Ala Gln Thr Trp Gly Gln 100105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly115 120 125 Gly Ser Gly Gly Ser Ala Leu Gln Ser Val Leu Thr Gln Pro ProSer 130 135 140 Val Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys SerGly Ser 145 150 155 160 Ser Ser Asp Met Gly Asn Tyr Ala Val Ser Trp TyrGln Gln Leu Pro 165 170 175 Gly Thr Ala Pro Lys Leu Leu Ile Tyr Glu AsnAsn Lys Arg Pro Ser 180 185 190 Gly Ile Pro Asp Arg Phe Ser Gly Ser LysSer Gly Thr Ser Ala Thr 195 200 205 Leu Gly Ile Thr Gly Leu Trp Pro GluAsp Glu Ala Asp Tyr Tyr Cys 210 215 220 Leu Ala Trp Asp Thr Ser Pro ArgAsn Val Phe Gly Gly Gly Thr Lys 225 230 235 240 Leu Thr Val Leu Gly AlaAla Ala His His His His His His Gly Ala 245 250 255 Ala Glu Gln Lys Leu260 7 20 PRT Homo sapiens 7 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro LeuAla Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly 20 8 15 PRT Homo sapiens 8Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 1 5 10 15 919 PRT Streptomyces cinnamoneus MISC_FEATURE (11)..(18) Xaa = Abu 9 AlaLys Gln Ala Ala Ala Phe Gly Pro Phe Xaa Phe Val Ala Asp Gly 1 5 10 15Asn Xaa Lys

What is claimed is:
 1. A method of inhibiting viral replication orinfection, comprising contacting a composition comprising a virallyinfected cell with an antibody, or antigen-binding fragment thereof,that binds to an aminophospholipid, in an amount effective to inhibitviral replication or infection.
 2. The method of claim 1, wherein saidantibody is a monoclonal antibody.
 3. The method of claim 1, whereinsaid antibody is an IgG antibody.
 4. The method of claim 1, wherein saidantibody is an antigen-binding fragment of an antibody.
 5. The method ofclaim 4, wherein said antibody is an scFv, Fv, Fab′, Fab, diabody,linear antibody, F(ab′)₂ antigen-binding fragment of an antibody or aCDR, univalent fragment, camelized or single domain antibody.
 6. Themethod of claim 1, wherein said antibody is a human, humanized orpart-human antibody or an antigen-binding fragment thereof.
 7. Themethod of claim 1, wherein said antibody is a chimeric, bispecific,recombinant or engineered antibody.
 8. The method of claim 1, whereinsaid antibody binds to phosphatidylethanolamine.
 9. The method of claim1, wherein said antibody binds to phosphatidylserine.
 10. The method ofclaim 1, wherein said antibody binds to phosphatidylserine andeffectively competes with the monoclonal antibody 3G4 (ATCC PTA 4545)for binding to phosphatidylserine.
 11. The method of claim 10, whereinsaid antibody is the monoclonal antibody 3G4 produced by hybridoma ATCCPTA
 4545. 12. The method of claim 1, wherein said virally infected cellis a mammalian cell.
 13. The method of claim 1, wherein said virallyinfected cell is a human cell.
 14. The method of claim 1, wherein saidvirally infected cell is infected with a hepatitis, influenza, HIV,herpes, paramyxovirus or arenavirus.
 15. The method of claim 1, whereinsaid method inhibits viral replication in said virally infected cell.16. The method of claim 1, wherein said method inhibits viral spreadfrom said virally infected cell.
 17. The method of claim 1, wherein saidvirally infected cell is located within an animal and said antibody isadministered to said animal.
 18. The method of claim 17, wherein saidanimal is a human patient.
 19. A method for treating an animal with aviral infection, comprising administering to said animal apharmaceutical composition comprising an antibody, or antigen-bindingfragment thereof, that binds to an aminophospholipid, in an amounteffective to inhibit viral replication or spread in said animal, therebytreating said viral infection.
 20. The method of claim 19, wherein saidpharmaceutical composition is administered to said animal intravenously.21. The method of claim 19, wherein said pharmaceutical composition isadministered to said animal as an aerosol.
 22. The method of claim 19,wherein said animal has, or is at risk for developing, hepatitis,influenza, AIDS, viral pneumonia or respiratory disease or Lassa fever.23. The method of claim 19, wherein at least a second, distinctanti-viral agent is administered to said animal.
 24. The method of claim23, wherein said at least a second, distinct anti-viral agent is ananti-viral agent from Table G.
 25. The method of claim 19, wherein saidanimal is a human patient.